spp.) are becoming increasingly popular due to the rising awareness of the health benefits of consuming blueberry fruit, which include decreased risk of cardiovascular diseases, improved cognitive performance, and decrease in aging-related damage [1
]. Commercially important blueberry species include lowbush (Vaccinium angustifolium
Ait.) and northern highbush (Vaccinium corymbosum
L.) mainly cultivated in the northern parts of the United States, and rabbiteye (V. virgatum
Ait.) and southern highbush (hybrids of V. corymbosum
, V. virgatum
, and V. darrowii
Camp.) grown mostly in the southern states [3
]. Recently, production of blueberries has expanded to 27 countries (in 2011) compared with only ten countries in 1990 [5
]. The United States is the largest producer of blueberries globally [5
], supplying 347.7 million kg of cultivated and wild blueberries in 2016 [6
]. The United States also plays an important role in the import and export trade of blueberries [7
]. In 2016, the United States exported 31.7 million kg of fresh and 25.4 million kg of frozen blueberries and imported 149 million kg of fresh and 75.6 million kg of frozen fruit [8
As global production and trade continues to rise, it becomes increasingly important to maintain fruit quality, nutrient content, phytosanitary safety, and eliminate pests and diseases in blueberries during storage to ensure that this fast-growing export and import market is not negatively impacted. Postharvest losses in fruits can vary from 10 to 40% [9
]. After harvest, blueberries have a shelf-life of approximately 7 to 40 days depending on the genotype, method of harvest, and storage regime [9
]. During postharvest storage, blueberry fruit quality can decline due to fruit softening [11
]. Other contributing factors in loss of fruit quality are postharvest diseases caused primarily by fungal plant pathogens such as Colletotrichum
spp. (ripe rot), Alternaria
spp. (Alternaria fruit rot), and Botrytis cinerea
(gray mold), among others [12
]. In addition to postharvest disease-causing organisms, it is important to eliminate foodborne pathogens or associated indicator organisms [16
]. Although outbreaks of foodborne illnesses associated with consumption of blueberry fruit have been relatively rare, produce brokers and buyers have begun to apply rigid (and typically proprietary) microbial standards to frozen blueberries destined for the processing market [19
]. Although similar standards currently are not in place for the fresh-market, reducing microbial risk remains a key consideration for fresh-market production as well [20
]. Finally, in order to export blueberries to other countries, they are required to be certified free of certain insect pests such as Mediterranean fruit fly (Ceratitis capitata
), South American fruit fly (Anastrepha fraterculus
), European grapevine moth (Lobesia botrana
), blueberry maggot (Rhagoletis mendax
), and plum curculio (Conotrachelus nenuphar
Fumigation of export goods with methyl bromide was the most commonly used phytosanitary treatment for elimination of pests, but has been phased out in the United States, with the exception of a few critical uses [23
]. Methyl bromide also requires the produce temperature to be increased in order to be effective, thereby breaking the cold-chain and potentially having an adverse effect on quality. Interruption of cold-chain can decrease shelf-life considerably by increasing undesirable fruit metabolism [25
]. Irradiation using gamma rays, X-rays, or electron beams could be an alternative to fumigation in eliminating pests and in preserving quality by reducing decay organisms and plant and human pathogens [23
]. Previous work supported the use of electron beam and gamma irradiation to maintain shelf-life and fruit quality attributes in blueberry fruit [27
]. In the United States, regulatory approval has been obtained for the use of irradiation on fresh fruits and vegetables up to 1 kGy [31
]. Previous studies suggested an irradiation dose of 0.4 kGy to be effective against most insect pests, 0.2–0.8 kGy to cause a 1-log reduction in surface bacterial pathogens causing foodborne illness, and higher doses of 1–3 kGy for postharvest disease-causing fungi [22
The objective of this study was to determine the effect of irradiating postharvest blueberry fruit using a new form of electron beam technology, Electronic Cold-PasteurizationTM (ECPTM) developed by ScanTech Sciences (Norcross, GA, USA) at their Research and Development (R&D) facility at Idaho State University (ISU). This R&D facility is a small-scale version of a commercial ECPTM food treatment facility, which is currently being constructed by ScanTech in McAllen, TX and will be operational in the fourth quarter of 2018. This technology employs a highly focused beam of electrons, treating samples for only milliseconds on a high-speed conveyor while maintaining cold-chain integrity. A key advantage of electron beam irradiation over gamma rays (from nuclear sources such as Cobalt-60) or X-rays is the ability to deliver extremely high dose rates with improved accuracy since the beam dynamics can be more precisely controlled. These high dose rates equate to significantly less time for treatment and, consequently, potential for higher quality produce. The ECPTM treatment can treat an entire truckload (around 60,000 clamshells) of blueberries in a little over 30 min, whereas gamma rays can take several hours for the same quantity (C. Starns, unpublished observations). This is the first study to investigate the effect of irradiation on fruit quality attributes, postharvest disease incidence, and surface microbes of food safety concern in two southern highbush blueberry cultivars treated with ECPTM prior to cold storage.
2. Materials and Methods
2.1. Fruit Collection and Irradiation
Two trials were conducted with hand-harvested fruit from southern highbush blueberry cultivars ‘Farthing’ and ‘Rebel’ in Alma, GA. In trial 1 (April 2016), ‘Farthing’ fruit were obtained from a commercial packing facility, where fruit had already been prepacked into pint-size clamshell containers (473 mL). In trial 2 (May 2016), ‘Rebel’ fruit were obtained from a different packing facility, also already prepacked in pint-size clamshells. In addition, trial 2 included ‘Farthing’ fruit hand-harvested by the investigators from a commercial blueberry farm and packed into pint-size clamshells.
A subsample of clamshells in each trial was taken directly to the University of Georgia, Athens, GA, USA (330-km transit in refrigerated cooler) to serve as an unshipped control (not transported to and from the irradiation facility). Initial fruit quality attributes and postharvest disease incidence were recorded from this unshipped control. The remaining fruit in clamshells were arranged on standard flats (12 clamshells/flat), placed in a styrofoam cooler with ice packs, and shipped overnight from Alma, GA to ISU, Pocatello, ID. A foam sheet was placed on the inner side of the lid of each clamshell and in between clamshells to minimize fruit injury during shipment.
At ISU, fruit in clamshells were subjected to electron beam irradiation treatment at ScanTech’s R&D facility using proprietary ECPTM technology. A 10-MeV electron beam, driven by an advanced high-energy electron accelerator, is magnetically focused through a scanning horn which delivers precision dose control. At the R&D facility, clamshells containing fruit were subjected to four levels of irradiation, 0, 0.15, 0.5, and 1.0 kGy; the treatments were completed in less than a second per clamshell. The respective doses were achieved using the National Institute of Standards and Technology (NIST)-traceable alanine pellets with extensive dose mapping on various blueberry configurations prior to the experimental fruit being shipped to the facility. Hundreds of data points were obtained and measured on a Bruker Bio-spin Electron Paramagnetic Resonance spectrometer, all of which are NIST traceable and International Organization for Standardization/American Section of the International Association for Testing Materials compliant. Treatments were replicated four times (i.e., four clamshells/irradiation level/postharvest storage period/cultivar), with a few exceptions where fewer replicate clamshells were available. The 0-kGy treatment served as an untreated control wherein fruit were shipped but not irradiated. After irradiation, fruit were shipped back by overnight courier to the University of Georgia where they were placed in a walk-in cooler at 2 to 4 °C under high relative humidity (>85%) until further assessment. The entire shipping and treatment process (from Alma to the treatment facility at ISU and to Athens for cold-storage and evaluation) took between 6 to 7 days. The unshipped control clamshells were stored in a 2 to 4 °C walk-in cooler until further evaluation. Fruit were removed from cold storage and evaluated for postharvest fruit quality attributes at 6, 13, and 25 days after irradiation treatment; microbial load on the fruit surface at 6 days after treatment; and postharvest disease incidence at 6 and 13 days after treatment followed by 4 days at room temperature. Fruit quality, microbial load and postharvest disease incidence analyses at a given time-point were performed using four replicates; for every replicate, fruit from a separate clamshell were used and divided for the above analyses.
2.2. Evaluation of Fruit Quality Attributes
For evaluation of fruit quality, visual assessment as well as measurement of fruit weight, texture, titratable acidity (TA), and total soluble solids (TSS) content were performed. For visual assessment, 30 fruit per replicate were scored for symptoms of bruising such as tears, dents, leakiness, or signs of mold. Fruit were examined by eye for visual defects and percent sound fruit were calculated. For fruit texture, two variables, fruit compression and skin puncture force, were measured on 12 fruit per replicate using a fruit texture analyzer (GS-15, Güss Manufacturing, Strand, South Africa); fruit were oriented on the equatorial plane for this assessment. For compression measurements, a 1.5-cm diameter plate was used with parameters set at forward speed 6 mm/s, measure speed 5 mm/s, and measure distance 1.00 mm. For skin puncture force measurements, a 1.5-mm flat-tip probe was used with parameters set at a forward speed 10 mm/s, measure speed 5 mm/s, and measure distance 3.00 mm. Fruit weight was recorded on 20 individual fruit per replicate using a balance (Quintix Precision Balance, Sartorius, Bohemia, NY, USA).
For TA and TSS measurement, juice was extracted from ~40 g of fruit per replicate using a household blender and centrifuged for 10 min at 3901X g using a benchtop centrifuge (Allegra X-22, Beckman Coulter Life Sciences, Indianapolis, IN, USA). The resulting supernatant was filtered through two layers of cheesecloth. To measure TSS, 300 μL of supernatant was tested using a digital handheld refractometer (Atago USA, Belleveue, WA, USA). For TA, the supernatant was titrated using an automatic mini titrator (Hanna Instruments, Woonsocket, RI, USA) and values were reported as percent citric acid (CA). Statistical analysis (one-way analysis of variance for a completely randomized design) was performed separately for each trial and cultivar using JMP Pro 12 (SAS Institute, Cary, NC, USA). Means were separated using Tukey’s Honest Significant Difference (HSD) test (α = 0.05).
2.3. Evaluation of Fruit Surface Contaminants
Microbial loads on the fruit surface were determined 6 days after treatment following the protocol described in Mehra et al. [35
]. One 50-g fruit sample (~30 berries) per replicate was placed in a 0.5-L flask with 50 mL of sterile phosphate buffer (pH 7.2), and the flask was agitated on a wrist action shaker (Burrell, Pittsburg, PA, USA) at medium speed for 15 min. Aliquots of the wash buffer and 1:20 and 1:100 dilutions were plated in triplicate onto plate count agar (PCA), dichloran rose bengal chloramphenicol agar (DRBC), and Petrifilms (3M Microbiology, St. Paul, MN, USA) for enumeration of aerobic bacteria, total yeasts and molds, and E. coli
and coliforms, respectively. PCA and DRBC dishes were incubated at room temperature and evaluated after 3 and 5 days, respectively. Petrifilms were incubated at 35 °C and colony counts made after 2 days. Colony-forming units (CFU) per gram of fruit were log-transformed and subjected to one-way analysis of variance using PROC GLM in SAS version 9.4 (SAS Institute, Cary, NC, UAS) followed by means separation using Tukey’s test.
2.4. Assessment of Postharvest Disease
An initial postharvest disease assessment was made on the unshipped control following 4 days of storage at room temperature (23–25 °C) to allow latent infections to manifest themselves [35
]. Subsequently, on fruit subjected to ECPTM
treatment, fruit samples (60 berries per replicate) were removed from postharvest storage 6 days (trials 1 and 2) and 13 days (trial 1 only) after treatment, and similarly incubated at room temperature for 4 days. The 13-day assessment was not included in trial 2 as poor fruit quality of ‘Rebel’ in that trial resulted in near 100% decay after cold storage and subsequent room temperature incubation. For each assessment date and replicate, the number of fruit with symptoms and signs of postharvest decay was counted following examination of fruit samples with a stereo microscope. Fungal pathogens associated with diseased fruit were identified macroscopically and microscopically (utilizing both stereo- and compound microscopes) based on characteristic symptoms and signs [36
]. Based on the number of fruit with disease symptoms and pathogen signs, postharvest disease incidence was calculated and arcsine-square root transformed for analysis by one-way analysis of variance using PROC GLM followed by means separation using Tukey’s test.
The objective of this study was to determine the effect of ECPTM
on fruit quality attributes, surface microbial load, and postharvest diseases on two southern highbush cultivars. ECPTM
treatment resulted in a cultivar-specific response on fruit quality. In ‘Rebel’, ECPTM
had no effect on visual appearance, fruit firmness, and skin toughness. In ‘Farthing’, however, ECPTM
at 1.0 kGy, resulted in a reduction in fruit firmness and skin toughness but did not affect the visual appearance of the fruit, which was assessed based on the presence of bruises and defects such as leakiness or dents. The differential cultivar response to irradiation could be due to inherent differences in fruit firmness between the two cultivars. ‘Rebel’ was softer and had lower firmness and skin puncture force than ‘Farthing’. Thus, irradiation may not have decreased firmness further in ‘Rebel’. Similar results with differences in responses of blueberry cultivars varying in fruit texture have been observed using previous irradiation studies with various radiation sources [21
]. Cultivars with firmer texture were softened after irradiation, whereas the effect of irradiation on two softer-textured cultivars varied; irradiation further softened fruit of one of the cultivars but had no effect on the other [38
]. These data indicate that fruit having inherently firmer texture may be softened by irradiation, whereas the texture of fruit with lower fruit firmness may not be affected.
In this study, fruit softening and a decrease in skin toughness in ‘Farthing’ occurred only at the highest irradiation dose of 1.0 kGy. These results are consistent with other studies that report a dose-dependent response to irradiation with higher doses resulting in a decrease in firmness in blueberry fruit regardless of the method of irradiation. When conventional electron beam irradiation was used to treat blueberries, doses of 1.1 kGy and higher affected fruit texture resulted in softening [28
]. Other studies using gamma irradiation around 0.75 kGy and higher reported increased softening in blueberries [21
]. The effect of higher doses of irradiation on fruit softening has also been observed with other fruits such as raspberries [40
], peaches [23
], apricots [23
], and grapes [43
In spite of changes in fruit firmness, irradiation did not change other fruit quality attributes such as total soluble solids content, titratable acidity, and weight. Apart from a few minor differences, our results are consistent with other studies that indicate no effect of irradiation on fruit quality characteristics related to flavor [21
]. The overall effect of irradiation on fruit firmness and quality in terms of consumer acceptability is an important consideration. In this study we did not perform sensory evaluations; only few other studies have conducted post-irradiation sensory analyses, and have shown mixed results related to irradiation induced softening and consumer acceptability [21
] in peaches and blueberries.
In addition to fruit quality attributes, it is important to understand the effect of irradiation on the presence of fruit surface organisms that may cause foodborne illness. Blueberries are produced in open fields and can harbor various human pathogens by route of animal waste, irrigation water, and handling by farm workers. After harvest, blueberries for the fresh market are not washed nor treated for surface pathogens [20
]. Therefore, it would be an added benefit if irradiation could reduce or eliminate such surface organisms. ECPTM
treatment was effective in reducing surface microbial load in both ‘Rebel’ and ‘Farthing’. In ‘Rebel’ irradiation at smaller doses was more effective in reducing surface pathogen load than in ‘Farthing’. This was likely because ‘Rebel’ harbored a higher load of microbes on the fruit surface than ‘Farthing’. In ‘Rebel’, aerobic bacteria and yeasts were reduced by 0.6–0.7 log units and coliforms by 2 log units at 1.0 kGy irradiation. In ‘Farthing’, similar reductions were observed for aerobic bacteria and yeasts, but not for coliforms. These results are partially consistent with previous studies suggesting irradiation doses between 0.2–0.8 kGy are sufficient to cause a 1-log reduction in surface bacterial pathogens such as E. coli
, and Listeria
]. In another study with blueberries, 0.4-kGy irradiation resulted in a 1-log reduction in Salmonella
], but those specific taxa were not investigated in the present study. The authors concluded, and we concur, that this level of reduction may reduce risk but not guarantee safety.
Blueberries are affected by various postharvest diseases caused mainly by plant-pathogenic fungi [21
]. In this study, some of the common postharvest pathogens B. cinerea
spp., as well as Aurebasidium
, and Cladosporium
were identified after postharvest storage. However, in our study ECPTM
treatment did not affect the incidence of symptoms and signs associated with postharvest pathogens. Compared with microbes located on the fruit surface, a much higher dose of irradiation, typically at 1–3 kGy, is necessary to eliminate plant-pathogenic fungi [32
]. Further, sensitivity of irradiation also can differ among various plant pathogens. Using an in vitro assay, inactivation of B. cinerea
, Penicillium expansum
, and Rhizopus stolonifer
was observed at irradiation doses of 3–4 kGy and 1–2 kGy, respectively [47
]. The maximum dose of irradiation of 1.0 kGy in our study may not have been sufficient to decrease postharvest decay pathogens. In addition, ‘Farthing’ had an inherently low prevalence of postharvest pathogens; hence, irradiation did not further reduce postharvest disease incidence.
Data from this study with the new ECPTM
approach is in agreement with previous research which recommends a dose between 0.5 and 1.0 kGy for blueberry fruit to avoid undesirable effects on fruit quality [21
]. While irradiation at this dose may provide protection from insect pests (not tested in this study) and some reduction in surface microbial load, more research is needed on its potential to reduce postharvest rots. In apples, mangoes, peaches, and carrots, irradiation combined with other postharvest treatments, such as cold, heat, fungicides, CaCl2
treatment, or modified atmosphere offered greater benefits in controlling postharvest diseases and maintaining higher fruit quality [48
]. Importantly, the above studies demonstrate that lower doses of irradiation are more effective when used in combination with other treatments than using irradiation alone. Blueberries are generally not treated after harvest, therefore future studies should focus on preharvest applications such as fungicides or calcium treatments in combination with irradiation and storage with modified atmosphere.
ECPTM is attractive because the method’s high dose rates allow the desired irradiation dose to be obtained in a considerably shorter period of time, reducing treatment bottlenecks during operation and potentially improving produce quality through shorter treatment times outside of the cold-chain. However, direct side-by-side comparisons of ECPTM with gamma rays or X-rays at identical irradiation doses (but varying dose rates as dictated by the method) have not been conducted previously, pointing to an important research need. Future research also should address one of the limitations of our study, the need to ship the fruit to and from the treatment facility after harvest and before postharvest storage, which could have impacted treatment efficacy.