Microbiology and Quality Attributes of ‘Pione’ Grapes Stored in Passive and Active MAP

Abstract

The quality of ‘Pione’ grapes was evaluated during passive and active modified atmosphere packaging (MAP) storage. In the passive MAP study, ‘Pione’ grapes were packaged in two types of films with an oxygen transmission rate (OTR) of either 440 mL/m²/d/atm (low OTR) or 1250 mL/m²/d/atm (high OTR) and stored at 25 °C or 10 °C. When the CO₂ concentration in low and high-OTR films stored at 25 °C reached 10% and 3%, respectively, on day 2, grape berries showed lower bacterial counts in the low-OTR films than in the high-OTR films. At 10 °C, the packages approached an equilibrium of 12% CO₂ in low-OTR films and 7% CO₂ in high-OTR films during 8 days of storage, and no difference was observed in the bacterial counts between the two films. In an active MAP study, ‘Pione’ grapes were stored in low-OTR (440 mL/m²/d/atm) and high-OTR (1170 mL/m²/d/atm) films flushed with air or high CO₂ (10%, 20%, and 30%) at 10 °C for 8 days. The CO₂ concentration in active MAP with low-OTR films reached approximately 20% by the end of storage, while that with high-OTR films approached an equilibrium of 10% CO₂ after 4 days of storage. The bacterial counts remained below the limit of detection until 4 days of storage in active MAP with high-OTR films. Although the fungal counts of berries were non-detectable or below the limit of detection in all active MAPs, Alternaria and Candida fungi and Chryseobacterium and Cutibacterium bacteria were found in the berries stored in active MAP. The firmness, soluble solid content, and surface color of the berries were not affected, regardless of the film type, in both passive and active MAP, and rachis browning due to high-CO₂ injury was not observed in any samples in active MAP. These results indicate that passive MAP with low-OTR films or active MAP of 10–20% CO₂ with high-OTR films at 10 °C were the optimum packing systems for ‘Pione’ grapes to control the physical and microbiological quality without high-CO₂ injury, such as rachis browning.

1. Introduction

The production of grapes has increased worldwide in the past decade [1,2], and it has become crucial to maintain the quality of grapes during postharvest handling. The quality of grapes deteriorates due to microbial spoilage, berry softening, and rachis browning during storage [3,4]. Gray mold, powdery mildew, or sour rot are common diseases of grapes caused by microbes. The infection with and proliferation of Botrytis cinerea particularly cause the development of gray mold and softening of berries. The common technique to suppress fungal proliferation is initial SO₂ fumigation and subsequent SO₂ release during transport [5,6]. However, excessive SO₂ can induce damage to rachis and berries, and reactions in people allergic to sulfite residues. As alternatives to SO₂ treatment, there have been studies on the effects of low-temperature storage [7], ethanol treatment [8], and high-CO₂ treatment [9] on controlling fungal growth.

Passive or active modified atmosphere packaging (MAP) is used as a storage method for reducing microbial growth in fresh and fresh-cut produce by the efficacy of a CO₂-enriched atmosphere, because CO₂ levels of >10% suppress the growth of both spoilage microorganisms and human pathogens [10]. Low-O₂ and high-CO₂ atmospheres within passive MAP are achieved by the natural interaction between the respiration of the packaged produce and transfer of gases through the packaging film, whereas active MAP with the application of the desired gas could be helpful in achieving the ideal equilibrium atmosphere readily and quickly [11]. High CO₂ in the packages can control microbial proliferation and also inhibit metabolic processes. With passive MAP, Artés-Hernández et al. [12] reported that an atmosphere inside films ranging from 4–14% O₂ and 8–21% CO₂ was effective in maintaining the quality of ‘Superior seedless’ grapes for 7 days at 0 °C followed by 4 days at 8 °C and 2 days at 20 °C. When table grapes were stored at 0 °C for 3 months followed by 20 °C for 3 days using passive MAP, browning was suppressed compared with conventional storage methods [4]. In an active MAP study on table grapes, MA storage with CO₂ concentrations of <10% was reported to be most effective at 5 °C, while storage in high-CO₂ atmospheres of >20% caused acetaldehyde and ethanol accumulation by inducing anaerobic respiration [13]. Cefola and Pace [14] also reported that table grapes packaged in active MAP with 10% CO₂ initially and stored at 2 °C effectively controlled the sensory and nutritional quality, whereas active MAP with initial 20% CO₂ accelerated acetaldehyde and ethanol accumulation during storage.

Although the storage quality of grapes would be maintained by optimal high-CO₂ atmospheres, the tolerance of grapes to CO₂ seems to be dependent on the CO₂ level, storage temperature, and cultivar. Therefore, it is necessary to consider the optimum CO₂ concentration for a target cultivar to avoid high-CO₂ injury and anaerobic respiration during MAP storage. In this study, we compared the effect of the gas concentration in the films on the microbiological and overall quality of ‘Pione’ grapes, a cultivar famous for its large-sized and black-colored berry, when stored in passive MAP at 25 °C and 10 °C and active MAP at 10 °C, and then determined the optimum packaging system for the storage and distribution of ‘Pione’ grapes. Storage temperatures of 25 °C and 10 °C were used in this study, because grapes are generally shipped at ambient temperature and the refrigerated shipping temperature for fresh produce is commonly 10 °C in Japan.

2. Materials and Methods

2.1. Fruit Materials

‘Pione’ grapes (Vitis labrusca L. × Vitis vinifera L.) were obtained from the Japan Agricultural Cooperative Group in Okayama Prefecture, Japan, in August 2020 for the passive MAP study and October 2020 for the active MAP study. Sixty-three clusters of grapes were transported to the laboratory at Kindai University at 10 °C, and the clusters were selected on the basis of size, color, firmness, and absence of defects or diseases.

2.2. Modified Atmosphere Packaging

For passive MAP, each individual grape cluster was packaged in two types of films with an oxygen transmission rate (OTR) of either 440 mL/m²/d/atm (low OTR) or 1250 mL/m²/d/atm (high OTR) (30 µm thick, 26 cm wide × 27 cm length; Sumitomo Bakelite, Tokyo, Japan), and then stored at 25 °C for 4 days or 10 °C for 8 days. For active MAP, individual clusters were placed in low-OTR (440 mL/m²/d/atm) and high-OTR (1170 mL/m²/d/atm) films, followed by flushing and evacuating three times with air, 10% CO₂, 15% CO₂, or 20% CO₂ before sealing. Three replicate packages of each treatment were stored at 10 °C for 8 days.

2.3. Gas Analysis in MA Packages

The O₂ and CO₂ concentrations in the packages were measured daily during the storage period using a gas chromatograph (GC-8AIT; Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector. The columns used for O₂ and CO₂ analysis were a Molecular Sieve 5A (60/80 mesh, 3.2 mm × 1.5 m; Shimadzu, Kyoto, Japan) at 60 °C and Porapak Q (60/80 mesh, 3.2 mm × 1.5 m; Shimadzu, Kyoto, Japan) at 90 °C, respectively.

2.4. Microbial Counts

‘Pione’ grape berries were taken on the initial, middle, and last days of the storage period to evaluate three replicate microbial counts. A 10 g sample (a berry) was homogenized in 90 mL sterile saline solution (0.85% NaCl in distilled water) in a sterile stomacher bag with a stomacher (Exnizer-400; ORGANO, Tokyo, Japan). Serial dilutions of each sample were produced in sterile saline solution. The microbial counts were enumerated using standard method agar (SMA) plates (Nissui Pharmaceutical, Tokyo, Japan) incubated at 37 °C for 48 h for mesophilic bacteria and potato dextrose agar (PDA) plates (Nissui Pharmaceutical) with 100 ppm chloramphenicol (Nacalai Tesque, Kyoto, Japan) incubated at 25 °C for 72 h for fungi. The bacterial and fungal counts were expressed as the log colony-forming units (CFU)/g.

2.5. Microbial Isolation and Identification

Bacteria and fungi were aseptically isolated from the homogenates of grape berries using SMA plates incubated at 37 °C for 48 h and PDA plates incubated at 25 °C for 72 h, respectively. Thirty-eight bacterial and fifteen fungal isolates were selected from different types of colonies growing on the respective plates from samples in active MAP storage.

Bacteria and fungi were identified to genus and species by the method described previously [15] with some modification. Briefly, bacterial and fungal genomic DNA was prepared from the respective isolates. The 1st 527 bp of the 16S rRNA gene or 323 bp of the 18S rRNA were amplified from the bacterial or fungal genomic DNA, respectively, by PCR (2720 Thermal Cycler; Applied Biosystems, Waltham, MA, USA). The PCR product was purified with illustraTM ExoProStarTM (GE Healthcare, Chicago, IL, USA) and then sequenced with a set of proprietary primers. The sequences were determined by electrophoresis with a DNA Sequencer (Eurofins Genomics, Tokyo, Japan). The sequencing data were analyzed using the Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 19 January 2021). A cut off of the highest matching score with the sequence in the database was chosen for species identity.

2.6. Storage Quality

Grape samples stored in passive MAP at 25 °C and 10 °C and active MAP at 10 °C were taken on the initial, middle, and last day of the storage period for the evaluation of quality attributes, including firmness, soluble solid content, surface color, and rachis browning. The firmness (N) of the berries was measured by the force required to shear a berry with a cutter blade using a Compact Tabletop Tester (EZ-test, Shimadzu, Kyoto, Japan). The soluble solid content (°Brix) of berries was determined using a Pocket Refractometer (PAL-1; Atago, Tokyo, Japan). The surface color (C* value) of the flesh of the berries was measured with a Handy Colorimeter (NR-12A; Nippon Denshoku, Tokyo, Japan). For the determination of the firmness, soluble solid content, and surface color, nine berries were used in each treatment. The rachis browning was evaluated for 3 clusters in each treatment and expressed as a browning scale (0: green, 1: yellow–green, 2: yellow, and 3: brown).

2.7. Statistical Analysis

Statistically significant differences (p ≤ 0.05) were determined for microbial counts and quality attributes. The mean values of each parameter were compared using Tukey’s honestly significant difference method.

3. Results

3.1. Passive MAP

Grapes were stored at 25 °C for 4 days and 10 °C for 8 days in passive MAP using two types of packaging films with low and high OTR. At 25 °C, the O₂ concentration in both the low and high-OTR films decreased similarly from 19% to 4–8% within 4 days of storage (Figure 1A). The CO₂ concentration in the low and high-OTR films increased from 0% to 10% and 3% on day 2 and 36% and 14% on day 4, respectively. At 10 °C, the packages approached an equilibrium of 10% O₂ and 12% CO₂ in the low-OTR film and 12% O₂ and 7% CO₂ in the high-OTR film within 8 days of storage (Figure 1B).

The bacterial counts of grapes stored in passive MAP with different OTR films are shown in Figure 2. The number of mesophiles of berries stored at 25 °C increased faster than that of those stored at 10 °C from the levels below the limit of detection (2.4 log CFU/g) on the initial day. Under 25 °C storage, the samples showed 2.5 log CFU/g and 3.3 log CFU/g on day 2, and 3.7 log CFU/g and 3.3 log CFU/g on day 4 in low and high-OTR films, respectively (Figure 2A). The difference in bacterial counts between the two OTR films on day 2 coincided with the different CO₂ concentrations in the packages that became apparent after 2 days of storage (Figure 1A). This indicates that the 10% CO₂ accumulated in the low-OTR film inhibited the increase in the number of bacteria. On the other hand, the growth of bacteria in samples stored at 10 °C increased to 3.5 log CFU/g in both low and high-OTR films by 8 days of storage, and no difference was observed in the number of bacteria between the two films (Figure 2B).

The changes in the firmness of the grape berries during passive MAP storage are shown in Figure 3. The firmness of the berries was not affected by the film type and storage temperature. The berry firmness in both low and high-OTR films increased from 7.5 N to 10–12 N on day 2 at 25 °C and on day 4 at 10 °C. Thereafter, the firmness remained constant throughout 4 days of storage at 25 °C and 8 days of storage at 10 °C, except for the berries stored in high-OTR films at 10 °C, where the firmness decreased significantly.

The soluble solid content in grape berries during passive MAP storage was determined (Figure 4). The soluble solid content was relatively constant at 16 °Brix throughout the storage period of 4 days at 25 °C and 8 days at 10 °C, regardless of the film type.

3.2. Active MAP

Since the passive MAP study revealed that a high-CO₂ atmosphere of 10% inhibited bacterial growth, active MAP flushed with either 10%, 15%, or 20% CO₂ was carried out to evaluate the microbiological effect of high CO₂ and tolerances to high CO₂ for commercial usage. The concentrations of CO₂ in low-OTR films during active MAP storage at 10 °C are shown in Figure 5A. The CO₂ concentration increased during storage in all packages, except for active MAP of 20% CO₂, where the CO₂ concentration remained at the initial level of 23%. The CO₂ concentration in packages flushed with air, 10% CO₂, and 15% CO₂ increased to 11%, 18%, and 22%, respectively, by the end of storage. When grapes were stored in active MAP with high-OTR films at 10 °C, the CO₂ concentration in the packages flushed with air approached a 5% equilibrium after 4 days of storage (Figure 5B). Other packages flushed with high CO₂ (10%, 15%, and 20%) approached an equilibrium of 10% CO₂ after 4 days of storage.

The bacterial counts in berries during active MAP storage at 10 °C are shown in Figure 6. The initial counts were higher in low-OTR films (2.9 log CFU/g) than in high-OTR films (<2.4 log CFU/g). The reason for this may have been the result of the differences in the initial fruit quality, because the samples used for the low-OTR-film experiment were harvested in the late season of production. In active MAP with low-OTR films, the bacterial counts of berries in packages flushed with 20% CO₂ on day 4 and flushed with air on day 8 were below the detection level, and the variation (SE) in the counts between treatments was large (Figure 6A). In comparison, flushing with high CO₂ kept the bacterial count below the limit of detection until 4 days of storage, which then increased by the end of storage when stored in active MAP with high-OTR films (Figure 6B). The fungal counts of berries were not detectable or below the limit of detection in all active MAP with both low and high-OTR films throughout the storage period (data not shown).

Table 1. Microorganisms isolated from ‘Pione’ grapes before storage and after storage in MA packaging flushed with either air or high CO₂ (10%, 15%, and 20%) for 8 days at 10 °C.

Low OTR		High OTR
Bacteria	Fungi	Bacteria	Fungi
Chryseobacterium taeanense	Alternaria alternata	Chryseobacterium lineare	Alternaria alternata
Chryseobacterium taichungense	Alternaria doliconidium	Cutibacterium acnes	Alternaria doliconidium
Cutibacterium acnes	Candida carpophila	Pantoea vagans

shows the bacteria and fungi isolated from berries on the last day of active MAP storage at 10 °C. No major differences were found in the variety and diversity of bacterial and fungal flora between samples stored in packages flushed with either air or high CO₂. Berries in active MAP with low-OTR films consisted of two bacterial genera, Chryseobacterium and Cutibacterium, one mold genus, Alternaria, and one yeast genus, Candida. Berries in active MAP with high-OTR films contained Pantoea sp. in addition to the bacteria in the low-OTR films and only the mold genus Alternaria in fungi.

The firmness of berries stored in active MAP with low-OTR and high-OTR films at 10 °C is shown in Figure 7A,B, respectively. In low-OTR films, the firmness increased from 14 N on day 0 to 17 N on day 4 only in the grapes flushed with air, which resulted in higher berry firmness in the air packages than in the high-CO₂-flushing packages, although the reason is unknown. Thereafter, there were no differences in the firmness of the berries among the packages on day 8. The firmness of berries stored in active MAP with high-OTR films was 15 N on day 0, and it did not change in all packages throughout 8 days of storage.

The soluble solid content in berries was 17.5 °Brix and remained constant during 8 days of storage in active MAP with low-OTR films at 10 °C (Figure 8A), whereas the content of berries stored in active MAP with high-OTR films increased from 15 °Brix on day 0 to 17.5 °Brix on day 4, and no differences were found among packages on day 8 (Figure 8B).

The berry and rachis browning of ‘Pione’ grapes during active MAP storage was evaluated. Since the C* values on the flesh of berries were between 10 and 15 and did not change significantly in all packages during storage at 10 °C, flesh browning was not observed (data not shown). The grape rachis was visually scored on a scale of 0 to 3, with 0 = green and 3 = brown. The score of rachis browning ranged from 0 to 1 in active MAP with low-OTR films and 0 to 0.5 in active MAP with high-OTR films during storage, indicating that all samples did not reach the limit of marketability (score 2) (data not shown).

4. Discussion

The beneficial effects of passive MAP and active MAP technology to control the deterioration of fresh produce are well recognized. The packaging technique commercially available for grapes is passive MAP because of the cheap and easy method. When passive MAP and active MAP of grapes were compared, Cefola and Pace [14] reported that initial concentrations of 0.03% CO₂/20% O₂ (passive MAP), 10% CO₂/20% O₂ (10% CO₂ active MAP), and 20% CO₂/20% O₂ (20% CO₂ active MAP) changed to 8% CO₂/10% O₂, 15% CO₂/7% O₂, and 26% CO₂/3% O₂, respectively, in the packages after 14 days of storage at 2 °C. The 10% CO₂ active MAP showed the best nutritional and sensory quality of table grapes. In contrast, Costa et al. [16] reported that similar equilibrium gas concentrations were shown under passive MAP and active MAP (initial concentrations of 3% CO₂/5% O₂, 3% CO₂/10% O₂, and 3% CO₂/15% O₂) at 5 °C, and the active MAP did not significantly affect the gas evolution and shelf life prolongation of table grapes. Liguori et al. [17] stored minimally processed table grapes in passive MAP with microperforated polypropylene packages and active MAP of 20% CO₂/20% O₂ or 15% CO₂/5% O₂ with non-microperforated polypropylene packages at 5 °C for 28 days and an additional 6-day period at 15–18 °C to simulate shelf life. The active MAP showed an excessively high CO₂ concentration (30–60%) in packages and caused berry and rachis decay. Liguori et al. [18] also reported that the same active MAP conditions stimulated anaerobic respiration, hastened soluble sugar degradation, caused higher weight loss, and altered the sensory quality as compared to passive MAP. These previous studies indicated that the CO₂ concentrations in MAP were crucial to control the quality of grapes, irrespective of the difference in the MAP method.

In our study, the CO₂ concentration in MAP with low-OTR films flushed with 0.03%, 10%, 15%, and 20% CO₂ reached 11%, 18%, 22 %, and 23% CO₂, respectively, by the end of storage at 10 °C, while all high-CO₂ active MAP with high-OTR films approached an equilibrium of 10% CO₂ during storage at 10 °C. With respect to the relationship between the CO₂ concentrations and microbial counts, a 10% CO₂ concentration in passive MAP with low-OTR films and active MAP with high-OTR films showed a decrease in bacterial growth. This result agrees with previous reports on other fruits, such as strawberries [19], persimmons [20], and mangoes [15]. These MAPs also contributed to the maintenance of firmness, soluble solid content, and flesh color of berries without rachis browning throughout the storage period at 10 °C and 25 °C. Regarding the effect of MA on the quality of grapes, Liguori et al. [17,18] reported that no chemical and sensory changes occurred in table grapes during storage in passive MAP where the O₂ concentrations ranged between 20.5% and 19% and the CO₂ concentration accumulated to 3%, whereas active MAP with toxic levels of CO₂ and reduced tension of O₂ hastened quality loss, including weight loss, firmness, total soluble solids, and visual appearance. Therefore, the MAP recommended in our study helped in maintaining the quality of ‘Pione’ grapes by avoiding the MA conditions in an extreme CO₂ concentration range beyond which quality loss and injury occur.

Previous CA studies on grapes revealed both beneficial and detrimental effects of high-CO₂ atmospheres on quality during cold storage, depending on the CO₂ levels. The CAs of 10% CO₂ and 3, 6, or 12% O₂ for ‘Red Globe’ table grapes stored at 1 °C for 8 weeks [4] and 10–15% CO₂ and 3, 6, or 12% O₂ for ‘Thompson Seedless’ table grapes stored at 0 °C for 3 months [21] were effective in controlling decay caused by gray mold. The combination of 15% CO₂ with 5% O₂ for ‘Red Globe’ and ‘Thompson Seedless’ table grapes stored for 20 days at 0 °C followed by 4 days at 20 °C [22], and ‘Autumn Seedless’ table grapes stored for 60 days at 0 °C followed by 7 days at 15 °C [23] resulted in good control of fungal infection caused by B. cinerea comparable to the use of SO₂. However, CA with CO₂ atmospheres of 15–25% for ‘Red Globe’ table grapes [4] and ‘Thompson Seedless’ table grapes [21], and 30% for ‘Kyoho’ grapes [24] developed an off flavor, and rachis and berry browning. The microbiological and qualitative responses of table grapes to high CO₂ atmospheres were also essentially followed by MAP studies on ‘Italia’ table grapes [13,14] and ‘Red Globe’ table grapes [17,18].

In our study with ‘Pione’ grapes, the fungal pathogen B. cinerea was not found, and the commonly isolated microbes were phytopathogenic organisms, such as Pantoea and Alternaria, except for Cutibacterium, which could be derived from human skin. The frequently isolated organisms from grapes include Pseudomonas, Chryseobacterium, Flavobacterium, Alternaria, Metschnikowia, and Candida, which are phytopathogenic and/or soilborne organisms living in a plant–soil environment [25,26,27]. The microflora of a given grape is not necessarily the same as that of other grapes, because potential sources of microbial contamination for fruit are from soil, agricultural water, pesticide solutions, and packing shed equipment in different environments [28,29]. Since B. cinerea can also infect senescent leaves and flower parts in the field [30], the fungus may not have existed at sufficient levels to infect to ‘Pione’ grapes in its growing environment, or the fungus may have remained latent due to the short storage period.

In conclusion, the use of passive MAP with low-OTR films or active MAP of 10–20% CO₂ with high-OTR films resulted in the best atmosphere to control the microbial proliferation and to not cause high-CO₂ injury, such as rachis browning, of ‘Pione’ grapes stored at 10 °C. Therefore, selecting packaging films with suitable OTR is crucial to maintaining the quality of ‘Pione’ grapes.