Oxygen Concentration Affects Chlorophyll Fluorescence in ...

J. AMER. SOC. HORT. SCI. 128(4):603–607. 2003.

Oxygen Concentration Affects Chlorophyll Fluorescence in Chlorophyll-containing Fruit and Vegetables Robert K. Prange,1 John M. DeLong, and Peter A. Harrison Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main St., Kentville, NS B4N 1J5, Canada Jerry C. Leyte and Scott D. McLean Satlantic Inc., Richmond Terminal, Pier 9, 3481 North Marginal Road, Halifax, NS B3K 5X8, Canada ADDITIONAL INDEX WORDS. nondestructive sensor, physiological stress, Malus sylvestris var. domestica, Pyrus communis, Musa ×paradisiaca, Actinidia deliciosa, Mangifera indica, Persea americana, Brassica oleracea Capitata Group, Capsicum annuum Grossum Group, Lactuca sativa ABSTRACT. A new chlorophyll fluorescence (F) sensor system called FIRM (fluorescence interactive response monitor) was developed that measures F at low irradiance. This system can produce a theoretical estimate of Fo at zero irradiance for which we have coined a new fluorescence term, Fα. The ability of Fα to detect fruit and vegetable low-O2 stress was tested in short-term (4-day) studies on chlorophyll-containing fruit [apple (Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.), pear (Pyrus communis L.), banana (Musa ×paradisiaca L.), kiwifruit (Actinidia deliciosa C.S. Liang & A.R. Ferguson), mango (Mangifera indica L.), and avocado (Persea americana Mill.)] and vegetables (cabbage (Brassica oleracea L. Capitata Group), green pepper (Capsicum annuum L. Grossum Group), iceberg and romaine lettuce (Lactuca sativa L.)). In all of these fruit and vegetables, Fα was able to indicate the presence of low-O2 stress. As the O2 concentration dropped below threshold values of 0 to 1.4 kPa, depending on the product, the Fα value immediately and dramatically increased. At the end of the short-term study, O2 was increased above the threshold level, whereupon Fα returned to approximately prestressed values. A 9-month study was undertaken with ‘Summerland McIntoshʼ apple fruit to determine if storing the fruit at 0.9 kPa O2, the estimated low O2 threshold value determined from Fα, would benefit or damage fruit quality, compared with threshold + 0.3 kPa (1.2 kPa O2) and the lowest recommended CA (1.5 kPa O2). After 9 months, the threshold treatment (0.9 kPa) had the highest firmness, lowest concentration of fermentation volatiles (ethanol, acetaldehyde, ethyl acetate) and lowest total disorders. Sensory rating for off-flavor, flavor and preference indicated no discernible differences among the three treatments.

Chlorophyll fluorescence (F) and its potential postharvest applications have been well reviewed (DeEll et al., 1999). In a study of ‘Marshall McIntoshʼ apple, DeEll et al. (1995) reported that F can detect low-O2 and high-CO2 stress before the development of associated disorders. Prange et al. (1997), using ‘Elstarʼ apples, concluded that both Fv/Fm and Fo can be sensitive indicators of low-O2 conditions. More recently, Prange et al. (2002), using an experimental prototype system, which measured a larger surface area than previously possible on an hourly basis, reported the discovery of specific low-O2 concentrations at which Fo and Fv/Fm suddenly increase and decrease, respectively. Based on the observation that measurement of Fo alone was sufficient to detect a low-O2 concentration, a new chlorophyll fluorescence (F) sensor system called HarvestWatch using fluorescence interactive response monitor (FIRM) sensors was developed (Satlantic Inc., Halifax, N.S., Canada), which measures F at low irradiance. This system is a nonpulse amplitude modulated proprietary technology, which produces a theoretical estimate of Fo at zero irradiance for which we have coined a new fluorescence term, Fα. The FIRM sensor geometry is designed to illuminate and detect Fα from a wide surface area, increasing its ability to measure Fα simultaneously on more than one fruit or vegetable. This study consisted Received for publication 4 Apr. 2003. Accepted for publication 24 Apr. 2003. Contribution 2258, Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada. 1Corresponding author; e-mail [email protected].


of: a short-term (4 d) low-O2 treatment to determine the ability of Fα to detect a low-O2 threshold similar to that reported by Prange et al. (2002); and a long-term (9 month) experiment to determine if apples can be successfully stored near the low-O2 threshold as estimated by Fα.

Fig. 1. CA sample jar with a FIRM sensor placed outside the jar.

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Materials and Methods EFFECT OF SHORT-TERM LOW-O2 TREATMENTS ON Fα. Representative chlorophyll-containing fruit, e.g., apple, pear, banana, kiwifruit, mango, and avocado, and vegetables, e.g., cabbage, green pepper, and iceberg and romaine lettuce, were placed in clear 4-L airtight plastic jars and connected via Tygon (Saint-Gobain Performance Plastics Inc., Wayne, N.J.) tubing in the lid to an O2 and CO2 monitoring and control system (Fig. 1). The O2 concentration in each jar was measured using a paramagnetic O2 sensor. A control jar was kept at ambient O2 (21 kPa) while a treatment jar had O2 lowered at a controlled rate (0.5 kPa every 12 h) from 3 kPa to close to 0 kPa over 96 h (4 d). Outside each jar, a FIRM sensing unit recorded Fα measurements every hour (Fig. 1). Four replicate samples were established for each fruit and vegetable product. Fresh, healthy, mature, market-quality produce was purchased from various local markets for each replicate. All experiments were performed at ≈20 °C and all sample jars were enclosed in a plastic box along with the FIRM device to keep the products dark-adapted. Oxygen, CO2 and Fα readings were measured and recorded hourly throughout the duration of each set of experiments. EFFECT OF USING Fα-BASED LOW-O2 CONTROLLED ATMOSPHERE (CA) ON APPLE QUALITY AFTER 9 MONTHS STORAGE. Using the methodology described above, a threshold value of 0.9 kPa O2 was determined for ‘Summerland McIntoshʼ. Threshold value refers to the O2 concentration below which the Fα value increased, signalling the onset of low-O2 stress. To determine if apples can be successfully stored for up to 9 months near the low-O2 threshold as estimated by Fα, three CA treatments were conducted on ‘Summerland McIntoshʼ apples at 3 °C with 0 to 0.5 kPa CO2 and O2 concentrations of 1) threshold Fα (0.9 kPa O2); 2) threshold Fα + 0.3 kPa (1.2 kPa O2); and 3) lowest commercial recommendationʼ (1.5 kPa O2). The CO2 concentration was kept low in order to avoid any possible CO2 effects. Fruit from four growers were harvested at Streif index [firmness/(starch index × soluble solids content (SSC))] values of 2.28, 2.50, 2.55, and 2.67, which would indicate they were just past the optimum harvest window for long-term storage (DeLong et al., 1999). They were held in 0.67 m3 stainless steel CA cabinets, each with an air-tight acrylic lid and a water-trough seal. Each cabinet held 4 ventilated PVC baskets, one for each grower. Samples were taken from each PVC basket at 3, 6, and 9 months and after an additional 7 d in air at 20 °C (CA + 7 d shelf life) for firmness, SSC, total acidity (TA), fermentation volatiles (ethanol, acetaldehyde, ethyl acetate), and disorders (including rots). Firmness, SSC, TA and disorder measurements were taken on samples of 25 apples removed from each CA treatment and removal date. Firmness values were measured and recorded using a Fruit Quality Tester (FQT, Geo-Met Instruments Inc., New Minas, N.S., Canada). Juice SSC was determined with a hand-held refractometer (Atago Co., Tokyo) and TA was determined from the same 25-apple composite sample by titrating 2 mL of apple juice with 0.1 mol·L–1 sodium hydroxide and expressing as malic acid equivalents (g·L–1). For measurement of volatile emissions, 4 fruit were quartered after the 7-d shelf life at the 3-, 6-, and 9-month removals. For each treatment, 1 quarter per fruit was sealed in a 1-L mason jar fitted with a septum lid and held for 4 h of headspace equilibration. A 1-mL headspace sample was then extracted with a syringe and analyzed on a gas chromatograph (GC) (Varian Inc., Walnut Creek, Calif.) with a SupelcoWax 10 (30 m × 0.53 mm i.d., 1.0 µm coating thickness) column, using ultra-high purity helium (99.99%, 604

Praxair, Mississauga, Ont.) as a carrier gas at a flow rate of 9 mL·min–1. The volatiles were detected by flame ionization using helium as the makeup gas at a flow rate of 30 mL·min–1; air and hydrogen flow rates were 30 and 300 mL·min–1, respectively. The analysis was performed isothermally at 80 °C with the injection port and detector temperatures set at 200 °C. Volatile standards were generated by evaporating a known amount of authentic acetaldehyde, ethanol, and ethyl acetate from a piece of filter paper dropped into a 4.4-L glass jar fitted with a Mininert gas-tight sampling valve (Alltech Assoc., Deerfield, Ill.). Quantification was done by comparison of the GC response of the sample to that of the standard volatile compounds (Song et al., 2001). A sensory rating for flavor, off-flavor, and percent preference was determined using a random sample of 10 individuals from the Atlantic Food and Horticulture Research Centre. Participants were asked to taste samples of fruit from the three different treatments and rate each category, and then select their overall preference. Apple flavor was rated on a scale of 1 to 9 with 1 being the worst rating and 9 being the best. Off-flavor was rated on a scale of 1 to 5, with 1 = no off-flavor and 5 = severe off-flavor. Sensory preference was expressed as a percentage of tasters preferring each treatment at each removal time. Treatment means were separated by the Waller Duncan k ratio t test, where k = 100 approximates the P ≤ 0.05 level. Analysis of variance was performed with PROC GLM using SAS software (SAS Institute, 1994). Unless noted otherwise, only results significant at P ≤ 0.05 are discussed.

Fig. 2. (a) Effect of decreasing O2 concentration on apple Fα (●), compared with Fα in ambient air ( ). Measurements were taken over 96 h at 20 °C in darkness. (b) Scatter-plot of O2 concentration versus apple Fα (●).


Results and Discussion EFFECT OF SHORT-TERM LOW-O2 TREATMENTS ON Fα. All of the 10 fruit and vegetables exhibited no change in Fα unless the O2 was below a critical threshold concentration. The main difference among the samples was in the threshold O2 concentration at which it occurred. Therefore, instead of showing the 96-h time-course measurements of O2 and Fα of all four replicates of the six fruit and four vegetables, three examples are presented as illustrations, apple (Fig. 2), pear (Fig. 3) and cabbage (Fig. 4). In all three 96-h time-course measurements of O2 and Fα (Figs, 2a, 3a, and 4a), product held in the ambient O2 clearly showed no Fα increase whereas the Fα of the product in the O2 treatment jar responded to the change in O2 concentration. As the O2 declined, there was no change in Fα unless the O2 concentration was below a critical threshold concentration. Below this threshold O2 concentration, the Fα value changed inversely to the O2 concentration, i.e., as O2 declined or increased, Fα increased and declined, respectively. When the O2 concentration was increased above the threshold after 72 h (Figs. 2a, 3a, and 4a), the Fα value returned to prestress values. The time-course illustration for apple (Fig. 2) was typical of what was observed in all ten products except for cabbage (Fig. 4). The cabbage response indicated some evidence of hysteresis (Fig. 4a and b) in which the Fα did not decline completely to prestress values. This was not observed in the other nine fruit and vegetables. The pear example was included to illustrate the sensitivity of Fα

to small changes in O2 concentrations near the threshold which is clearly visible between 24 and 48 h (Fig. 3a). Using the scatter-plots, the O2 threshold can be estimated, e.g., ≈0.8 kPa (Fig. 2b), 0.8 kPa (Fig. 3b), and close to 0 kPa (Fig. 4b). The estimated O2 thresholds of all four replicates of the 10 fruit and vegetables (Table 1) illustrate the variation in O2 threshold within samples of the same product and amongst different products. The scatter-plot for pear (Fig. 3b) and cabbage (Fig. 4b) show the O2 concentration went below 0 kPa even though every effort was made to accurately calibrate the O2 sensor, which did not have a linear response to O2 concentration in the 0 to 0.5 kPa range. Since it was part of a gas sampling system which ran continuously, it was necessary to accurately calibrate it in the O2 range where most of the O2 thresholds appeared to occur, i.e., 0.5 to 1.5 kPa . As a consequence, it was less accurate as O2 concentration approached 0 kPa. Conversely, a productbased sensor, such as Fα, can signal low-O2 stress regardless of the O2 sensor accuracy. EFFECT OF USING Fα-BASED LOW-O2 CA ON APPLE QUALITY AFTER 3, 6, AND 9 MONTHS STORAGE. After 3, 6, and 9 months storage, there was no significant treatment effect on SSC and TA (data not shown). Although not significant, firmness retention after 3 and 6 months appeared to be highest in the Fα threshold treatment (0.9 kPa O2), followed by the Fα threshold + 0.3 kPa (1.2 kPa O2) and CA (1.5 kPa O2) treatments (data not shown). After 9 months storage, this trend was statistically significant (Fig. 5).

Fig. 3. (a) Effect of decreasing O2 concentration on pear Fα (●), compared with Fα in ambient air ( ). Measurements were taken over 96 h at 20 °C in darkness. (b) Scatter-plot of O2 concentration versus pear Fα (●).

Fig. 4. (a) Effect of decreasing O2 concentration on cabbage Fα (●), compared with Fα in ambient air ( ). Measurements were taken over 96 h at 20 °C in darkness. (b) Scatter-plot of O2 concentration versus cabbage Fα (●).


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Fig. 5. Apple firmness values at harvest, after 9 months storage at 3 °C and after an additional 7 d at 20 °C (shelf life). At harvest n = 4 (one sample from each grower) for each treatment and for 9 months and 9 months + 7 d shelf life n = 50 (25 from growers 1 and 2 only). Error bar represents SE of the mean.

After a 7-d shelf life, fruit firmness was lower in all three treatments but the same treatment effect was still present (Fig. 5). There were no low-O2 disorders except at the 6 month removal + 7 d shelf life, which had ≈4% in the 0.9 kPa O2 treatment. Total disorders (including rots) were highest after 9 months storage + 7 d shelf life but they were not significantly different among the three treatments, ranging from a low of 16% in the 0.9 kPa O2 treatment to 26% in the 1.2 kPa O2 and 20% in the 1.5 kPa O2 treatments. The disorders were senescent-related, e.g. senescent breakdown and vascular breakdown, rather than low-O2 disorders. All three fermentation-related volatiles (ethanol, acetaldehyde, ethyl acetate) were similar at 3 and 6 months (data not shown) but at 9 months, they increased in the 1.5 kPa O2 treatment, compared with the 1.2 kPa and 0.9 kPa O2 treatments (Fig. 6). It has been generally accepted that ethanol, the final product of fermentation, will increase as the O2 concentration is lowered to its threshold or below, resulting in the recent development of a CA control system based on this principle (Schouten et al., 1997; Veltman et al, 2003). In addition, various research articles have suggested a correlation (Prange et al., 1997) or a direct association (Toivonen and DeEll, 2001) between fluorescence changes

Fig. 6. Apple volatile headspace concentrations after 9 months storage at 3 °C and after an additional 7 d at 20 °C (shelf life). Headspace concentrations were measured in 4-L jars containing 100 g fresh weight fruit. Error bar represents SE of the mean.

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Table 1. Estimated O2 thresholds (kPa) of six fruit and four vegetables using scatter-plots of O2 versus Fα, as illustrated in Figs. 2b, 3b, and 4b, which were apple replication 1, pear replication 3, and cabbage replication 2, respectively. Note that if the O2 threshold estimate was less than 0.0 kPa, it was expressed as 0.0 kPa in the table below. Sample (replication) Product 1 2 3 4 Fruit Apple 0.8 0.7 0.8 0.8 Avocado 1.0 0.6 0.5 0.7 Banana 0.5 0.5 0.2 0.0 Kiwifruit 0.7 0.5 0.5 0.2 Mango 0.4 0.6 0.6 0.5 Pear 0.5 0.5 0.8 0.5 Vegetables Cabbage 0.4 0.0 0.4 1.0 Green pepper 1.4 1.0 0.5 0.7 Iceberg lettuce 0.0 0.0 0.0 0.0 Romaine lettuce 0.4 0.0 0.0 0.0

and ethanol content. However, in our long-term study, ethanol, acetaldehyde and ethyl acetate concentrations did not increase at the lower 0.9 kPa threshold O2. If there is a direct association between ethanol and changes in chlorophyll fluorescence, then the 1.5 kPa O2 treatment would be expected to have the highest Fα but this was not observed. This suggests that, at O2 concentrations above the threshold, ethanol concentration was not tightly linked to either O2 concentration or Fα values. Perhaps, when O2 was kept just above its threshold, fermentative metabolism, as measured by fermentation products such as ethanol, was not stimulated during long-term storage. Another possibility is that, after 9 months storage, the fruit in the 1.5 kPa O2 treatment were more senescent than in the other two lower O2 treatments and began to produce senescence-based fermentation volatiles. Further research is needed to explain the apparent absence of a relationship between O2 concentration, ethanol and Fα when O2 is kept above its threshold. The flavor and off-flavor rating and treatment preference varied with removal time and storage treatment without any pattern being discerned. The most preferred fruit appeared to be from the 0.9 kPa treatment at 3 months, the

Fig. 7. Sensory rating of apples after 3, 6 and 9 months storage at 3 °C and after an additional 7 d at 20 °C (shelf life). Flavor was rated on a scale of 1 to 9 with 1 being the worst rating and 9 being the best. Off-flavor was rated on a scale of 1 to 5 with 1 = no off-flavor and 5 = severe off-flavor. Preference was expressed as percent of tasters preferring each treatment at each removal time.


1.5 kPa treatment at 6 months with no treatment preference at 9 months (Fig. 7). From this study, we conclude that Fα, using the HarvestWatch system, responded to low-O2 in a manner similar to Fo, as reported previously by Prange et al. (1997, 2002). If the O2 concentration dropped below a threshold value, Fα responded in an inverse relationship to the O2 concentration. If the O2 concentration was raised above the threshold, Fα returned to prestress values, with a hysteresis effect sometimes evident. Fα responded to low-O2 conditions in a similar pattern in all the fruit and vegetables studied. Storing apples for up to 9 months at an O2 concentration based on the Fα threshold (0.9 kPa), which was lower than the lowest commercial CA recommendation of 1.5 kPa, did not cause low-O2 damage and improved firmness retention. Furthermore, the Fα values did not appear to lose accuracy near 0 kPa O2, compared with the O2 sensor, but this will need to be substantiated with more research. The Fα measurement may be used commercially to warn a CA operator that the lowest acceptable O2 concentration has been achieved, thereby optimizing conditions to yield higher quality, post-storage fruit and vegetables. Additional unpublished research suggests Fα can also detect other stresses, e.g. high CO2 in storage and drought in growing plants. Literature Cited DeEll, J.R., R.K. Prange, and D.P. Murr. 1995. Chlorophyll fluorescence as a potential indicator of controlled-atmosphere disorders in ‘Marshallʼ


McIntosh apples. HortScience 30:1084–1085. DeEll, J.R., O. van Kooten, R.K. Prange, and D.P. Murr. 1999. Applications of chlorophyll fluorescence techniques in postharvest physiology. Hort. Rev. 23:69–107. DeLong, J.M., R.K. Prange, P.A. Harrison, R.A. Schofield, and J.R. DeEll. 1999. Using the Streif Index as a final harvest window for controlledatmosphere storage of apples. HortScience 34:1251–1255. Prange, R.K., S.P. Schouten, and O. van Kooten. 1997. Chlorophyll fluorescence detects low oxygen stress in ‘Elstarʼ apples. Proc. 7th Intl. Controlled Atmosphere Res. Conf., Davis, Calif. vol. 2:57–64. Prange, R.K., J.M. Delong, J.C. Leyte, and P.A. Harrison. 2002. Oxygen concentration affects chlorophyll fluorescence in chlorophyll-containing fruit. Postharvest Biol.Technol. 24:201–205. SAS Institute. 1994. SAS/STAT userʼs guide. vols. 1–2. version 6. 4th ed. SAS Ins., Inc., Cary, N.C. Schouten, S.P., R.K. Prange, J.A. Verschoor, T.R. Lammers, and J. Oosterhaven.. 1997. Improvement of quality of ‘Elstarʼ apples by dynamic control of ULO conditions. Proc. 7th Intl. Controlled Atmosphere Res. Conf., Davis, Calif. vol. 2:71–78. Song, J., L. Fan, C.F. Forney, and M.A. Jordan. 2001. Using volatile emissions and chlorophyll fluorescence as indicators of heat injury in apples. J. Amer. Soc. Hort. Sci. 126:771–777. Toivonen, P.M.A. and J.R. DeEll. 2001. Chlorophyll fluorescence, fermentation product accumulation, and quality of stored broccoli in modified atmosphere packages and subsequent air storage. Postharvest Biol.Technol. 23:61–69. Veltman, R.H., J.A. Verschoor, and J.H. Ruijsch van Dugteren. 2003. Dynamic control system (DCS) for apples (Malus domestica Borkh. cv. ‘Elstarʼ): Optimal quality through storage based on product response. Postharvest Biol.Technol. 27:79–86.

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