Glozer Dr, Kitren

Ferguson, Louise

Krueger, William H

Burns, Jackie :

Smail, S.H. :

Anderson, M. :

Nunez, J. :

Valleza, D. :

Evaluation of antioxidants to reduce physical damage in â?? Manzanillo â?? olives in 2006 Kitren Glozer and Louise Ferguson , Plant Sciences Department , U . C . Davis William Krueger , Farm Advisor , Glenn County Jackie Burns , University of Florida S . - H . Smail , M . Anderson , J . Nunes , D . Valleza , Student assistants ( CSUChico ) Summary : Several antioxidants were tested for reduction of physical damage induced by containerized shaking of â?? Manzanillo â?? olives in 2006 . Antioxidants tested included glutathione ( naturally - occurring free radical scavenger ) , ascorbic acid , a proprietary lactic / acetic acid solution ( â?? an organic low Ph solution â?? ; M . Erickson , personal communication ) , and quercetin and luteolin , flavones found in olive that have demonstrated strong anti - oxidant properties . Antioxidants were applied either as sprays on single limb replicates prior to damage , or as immersion overnight after mechanical damage . Different methods of inducing mechanical damage were tested to reproduce as closely as possible the type of damage observed in mechanical harvest trials in 2006 . Ascorbic acid applied prior to hand harvest and mechanical damage improved the appearance of fruit after overnight storage by reducing the severity of bruising ; luteolin alone and in combination with ascorbic acid also decreased apparent damage . Problem and its significance : Mechanical harvest of olives results in varying levels of visible damage within hours of fruit removal . Damage becomes more visible over time as the fruit is exposed to air after harvest . Bruising consists of a local degradation of tissue combined with intracellular water exit ( free water ) and browning ( oxidation ) of phenolic compounds from released intracellular water . Candidates for testing antioxidant potential included several chosen from naturally - occurring plant antioxidants . Glutathione is possibly the most abundant antioxidant scavenger found in plant and animal cells ( Wonisch and Schaur , 2002 ) and glutathione is involved in quenching free radicals through the ascorbate / reduced glutathione cycle ( Alscher , 1989 ; Smirnoff , 1995 ) . L - ascorbic acid ( vitamin C ) is a well - known antioxidant and part of the same cycle . Luteolin and quercetin , flavones found in the polyphenolic fractions of olive oil ( Andrikopoulos et al . , 2002 ) are purported to have antioxidant activity two to four times greater than that of vitamin E ( Cao et al . , 1997 ) . A mixture of lactic and acetic acids â?? a proprietary solution used for storage of freshly - harvested olives prior to processing to prevent degradation of color â?? was also tested . Both spray treatments and immersion tests were used to simulate preharvest and postharvest treatments that might have potential in olive cultivation and processing in combination with mechanical harvest . Objectives : 1 . Investigate antioxidant potential for reduction of damage due to simulated mechanical harvest . 2 . Evaluate fruit appearance after treatment by spray vs immersion methods to determine whether pre - or postharvest application may be better . 3 . Design a mechanical damage system that induces mechanical damage similar to that observed with Korvan harvester for testing purposes . Plans and Procedures : The experiment was carried out at the Nichols Soils Lab in Arbuckle on six year old â?? Manzanillo â?? olive trees planted to a hedgerow system with a 12 ft . X 18 ft . spacing.and drip irrigated Spray treatments were laid out in a complete random design in which 5 shoots bearing numerous fruit were selected for each treatment among several similarly - cropped trees with fruit at similar stages of maturity ( few fruit colored past green - mature stage ) . Fruit were treated with sprays of antioxidants + Helena Premium MSO ( methylated seed oil ; 0.05 % v / v ) on 16 October , hand - harvested on 18 October and treated to mechanical damage by shaking in containers . Controls included untreated , unshaken fruit , untreated , shaken fruit , adjuvant only spray ( in the case of spray treatments ) and water immersion ( in the case of immersion treatments ) . Sprays were applied to drip by hand - held spray bottles . Antioxidants included L - ascorbic acid ( vitamin C ) and luteolin ( Table 1 ) ; a combination treatment included ascorbic acid followed by luteolin . All antioxidant treatments were in aqueous solution although luteolin was solubilized initially in absolute ethanol ( solubility is 5 mg luteolin per ml ethanol ) . Antioxidants were obtained either from Sigma - Aldrich or vitacost.com ( quercetin and glutathione ) ; the lactic / acetic acid solution was donated by its manufacturer . All fruit from treated shoots were harvested and subjected to mechanical damage after transport to UC Davis . . Mechanical damage methods were tested to produce similar internal and external bruising as seen in fruit mechanically harvested by the Korvan harvester . Fruit damage was most similar when produced by shaking sampled fruit as a batch sample in a large covered plastic jar for 7 seconds . An overnight storage period at ambient temperature followed shaking . In the case of sprayed fruit , storage was in paper bags open to the air . Immersion tests with antioxidants after induced mechanical damage were also conducted to simulate postharvest submersion . Treatments included a water control , ascorbic acid , glutathione , ascorbic acid + glutathione , quercetin and a proprietary lactic / acetic acid solution ( Table 1 ) . Immersion was overnight . Each treatment was replicated on fruit collected from 5 shoots randomly distributed among the spray treatment shoots . Mechanical damage was induced immediately after harvest in samples collected for immersion tests . Harvested fruit were placed in net bags , immersed in the field and kept completely submerged in solutions overnight at ambient temperature . Fruit evaluation was conducted 19 October on a subsample of 25 fruit from each treated shoot selected at random from each replicate . Each fruit was individually numbered and scores for the appearance of external and internal bruising was recorded for each fruit individually . Internal bruising was evaluated when fruit were cut longitudinally along the seed and both halves were evaluated as a total sample . Evaluation consisted of rating fruit visually as : 1 . Externally bruised ( 1 , 2 , 3 scale with 3 most bruised ) 2 . Internally bruised ( 1 , 2 , 3 scale with 3 most bruised ) Analyses of variance were performed with Proc GLM procedure of SAS ( SAS Institute Inc . , Cary , NC ) and mean separations were tested by Duncan = s Multiple Range Test ; P = 0.05 . Spray treatments were compared without immersion tests ; immersion treatments were also compared without spray treatments . All treatments were also compared for significant differences . Results : Spray treatment comparison ( Table 2 ) External bruising was least in the untreated , unshaken control . Among treatments subjected to mechanical damage ( shaking ) , the untreated , shaken control showed the most damage . All antioxidant treatments significantly decreased the appearance of external bruising to the some extent . Internal bruising was also least in the untreated , unshaken control . The appearance of internal bruising was greatest in the untreated , shaken control , adjuvant control and the luteolin treatment . Internal bruising was decreased significantly by ascorbic acid and ascorbic acid followed by luteolin . Immersion treatment comparison ( Table 3 ) External bruising was least in the shaken , water control and the ascorbic acid + glutathione treatment . The lactic / acetic acid treatment showed the greatest amount of external bruising ; every impact bruise was clearly marked after this treatment . All other treatments were significantly different and intermediate to the above treatments . Internal bruising was also least in the shaken , water control , greatest in the ascorbic acid treatment , and significantly different and intermediate to these treatments for other antioxidant immersion treatments . Spray and immersion comparison ( Table 4 ) When all treatments were compared , external bruising was least in the untreated , unshaken control with all other treatments significantly greater . The lactic / acetic acid treatment showed the greatest external bruising . Sprays of antioxidants were all significantly better in reducing the effects of external bruising than were the immersion treatments , with the exception of the ascorbic acid + glutathione immersion treatment . This treatment , however , was not as good as sprays with luteolin or ascorbic acid + luteolin . Sprays of adjuvant , ascorbic acid , ascorbic acid + luteolin gave the best reduction in internal bruising and were better than all immersion treatments with the exception of the luteolin spray alone . Conclusions and considerations for future work : Spray treatments of antioxidants were generally better than immersion in reducing external and internal bruising due to mechanical damage , although not all antioxidants tested in sprays or immersions were duplicated in the other form of application . The treatments most effective in reducing both forms of bruising were sprays that included the methylated seed oil adjuvant alone and in combination with ascorbic acid and / or luteolin . Mechanical damage induced by shaking proved a viable substitute for mechanical harvest for testing purposes . While many other antioxidants may be tested , including those that are oil - based ( such as α - tocopherol â?? vitamin E ) , those that were tested are readily solubilized in water . Solubility of native α - tocopherol was found to be extremely poor in aqueous solution ; a water - soluble form exists for diagnostic purposes ( Trolox ) , however , this form is prohibitively expensive and was not tested . Of those tested , ascorbic acid is very inexpensive and available as food - grade . This may prove to be a good treatment to test preharvest and prior to application of chemical loosening agents and mechanical harvest . Efficacy may be increased by other adjuvants that encourage better penetration . Other antioxidant treatments that may be tested in future could include ethoxyquin or diphenylamine , used as postharvest antioxidant drenches or sprays in Canada with some apple varieties in which scald can be a problem ( Kupferman and Guzwiler , 2003 ) . We propose testing ascorbic acid spray applications as a preharvest treatment in fall , 2007 , incorporating this treatment into our mechanical harvest trial . Fruit would be collected for evaluation postharvest and subsampled for processing and evaluation after processing . Acknowledgements : We appreciate the use of the Nichols Soils Lab facility and the support of the California Olive Commission and California State University Chico in funding research . Pertinent literature : Alscher RG 1989 Biosynthesis and antioxidant function of glutathione in plants . Physiol . Plant . 77 : 457 - 464 . Andrikopoulos , N.K . , A.C . Kaliora , A.N . Assimopoulou , and V.P . Papageorgiou . 2002 . Inhibitory activity of minor polyphenolic and nonpolyphenolic constituents of olive oil against in vitro low - density lipoprotein oxidation . J . Med . Food 1 : 1 - 7 . Burns JK , Buker RS , Roka FM ( 2005 ) Mechanical harvesting capacity in sweet orange is increased with an abscission agent . Horttechnology 15 : 758 - 765 . Cao , G . , E . Sofic , and R.L . Prior . 1997 . Antioxidant and prooxidant behavior of flavonoids : Structure - activity relationships . Free Rad . Bio . Med . 22 : 749 - 760 . Kupferman , E . and J . Gutzwiler . 2003 . Use of diphenylamine , ethoxyquin and Semperfresh on Anjou pears . http : / / postharvest.tfrec.wsu.edu / EMK2002G.pdf . Liebler , D.C . , D.S . Kling And D.J . Reed . 1986 . Antioxidant Protection Of Phospholipid Bilayers By A - Tocopherol : control of a - tocopherol status and lipid peroxidation by ascorbic acid and Glutathione . J . Biol . Chem . 261 : 12114 - 12119 . Silva , M.M . , M.R . Santos , G . Caroço , , R . Rocha , G . Justino , and L.Mira . 2002 . Structure - antioxidant activity relationships of flavonoids : A Re - examination . Free Rad . Res . 36 : 1219 - 1227 . Smirnoff N 1995 Antioxidant systems and plant response to the environment . In " Environment and Plant Metabolism : Flexibility and Acclimation " ( N Smirnoff ed ) , Bios Scientific , Oxford , pp . 217 - 243 . Wonisch , W . and R.J . Schaur . 2002 . Chemistry of glutathione . In : Significance of Glutathione to Plant Adaptation to the Environment , vol . 2 ( D . Grill , M . Tausz , and L . J . De Kok , eds ) . Springer , 280 p . Yang , B . , A . Kotani , K . Arai , and F . Kusu . 2001 . Estimation of the antioxidant activity of flavanoids from their oxidation potentials . Analy . Sci . 17 : 599 - 604 . Table 1 . Anitoxidant treatments applied to â?? Manzanillo â?? olive in 2006 : effects on mechanical x . damage induced by shaking Spray treatments , 16 Oct Immersion treatments , 18 Oct Untreated , unshaken control Untreated , shaken control Water control 0.05 % Helena Premium MSO control 1000 ppm ascorbic acid 1000 ppm L - ascorbic acid + Premium MSO 10 ppm glutathione 10 ppm luteolin + Premium MSO Ascorbic acid + glutathione 10 ppm quercetin Ascorbic acid , followed by luteolin ( both Lactic / acetic acid ( proprietary solution , with Premium MSO ) concentrations unknown ) x Fruit subjected to shaking as a mass sample in a large plastic jar to simulate damage received by mechanical harvester . Treatments applied by hand - held spray bottle or by immersion . Fruit treated with sprays were harvested 2 days post - treatment , shaken after harvest and evaluated 24 hr after storage at ambient temperature . Fruit treated by immersion were harvested , shaken prior to immersion overnight , then evaluated . All fruit evaluated 19 Oct . Table 2 . Comparison of spray treatments with antioxidants and their effect on mechanical damage appearance in â?? Manzanillo â?? olive in 2006 . y Bruising Treatment External Internal x Untreated , unshaken control 0.02 c 0.03 d Untreated , shaken control 1.20 a 1.76 a 0.05 % Helena Premium MSO control 1.06 b 1.58 b 1000 ppm L - ascorbic acid + Premium MSO 1.05 b 1.46 c 10 ppm luteolin + Premium MSO 0.97 b 1.66 ab Ascorbic acid , followed by luteolin ( both with 1.00 b 1.45 c Premium MSO ) x Mean separation within columns by Duncanâ??s Multiple Range Test , P = 0.05 . y External and internal bruising rating system ( 1 , 2 , 3 scale with 3 most bruised ) . Table 3 . Comparison of immersion treatments with antioxidants and their effect on mechanical damage appearance in â?? Manzanillo â?? olive in 2006 . y Bruising Treatment External Internal x Water control 1.22 d 1.73 c 1000 ppm L - ascorbic acid 1.50 c 2.32 a 10 ppm glutathione 1.78 b 2.01 b Ascorbic acid + glutathione 1.18 d 1.97 b 10 ppm quercetin 1.46 c 2.03 b Lactic / acetic acid ( proprietary solution , 2.34 a 1.90 b concentrations unknown ) x Mean separation within columns by Duncanâ??s Multiple Range Test , P = 0.05 . y External and internal bruising rating system ( 1 , 2 , 3 scale with 3 most bruised ) . Table 4 . Comparison of spray and immersion treatments with antioxidants and their effect on mechanical damage appearance in â?? Manzanillo â?? olive in 2006 . y Bruising Treatment External Internal x Untreated , unshaken control 0.02 g 0.03 g Untreated , shaken control 1.20 d 1.76 cd 0.05 % Helena Premium MSO control 1.06 ef 1.58 ef 1000 ppm L - ascorbic acid + Premium MSO 1.05 ef 1.46 f 10 ppm luteolin + Premium MSO 0.97 f 1.66 de Ascorbic acid , followed by luteolin ( both with 1.00 f 1.45 f Premium MSO ) Water control 1.22 d 1.73 d 1000 ppm L - ascorbic acid 1.50 c 2.32 a 10 ppm glutathione 1.78 b 2.01 b Ascorbic acid + glutathione 1.18 de 1.97 b 10 ppm quercetin 1.46 c 2.03 b Lactic / acetic acid ( proprietary solution , 2.34 a 1.90 bc concentrations unknown ) x Mean separation within columns by Duncanâ??s Multiple Range Test , P = 0.05 . y External and internal bruising rating system ( 1 , 2 , 3 scale with 3 most bruised ) .