Comparison of three storage techniques for post-harvest quality preservation of six commercially available cultivars of apple

ABSTRACT

To evaluate the influence of 1-methylcyclopropene (1-MCP) treatment and ultra-low oxygen storage systems (ULO 1 2.0 kPa CO₂ and 1.0 kPa O₂) and (ULO 2 2.5 kPa CO₂ and 1.5 kPa O₂) on the chemical composition, sensory quality and proliferation intensity of microorganisms during long-term storage of apple, a two-year study was conducted, using two autumn cultivars: ‘Auksis’, ‘Orlik’, and four winter cultivars: ‘Antej’, ‘Belorusskoje Malinovoje’, ‘Sinap Orlovskij’ and ‘Zarja Alatau’, grown in cool climate. Fruits were stored for 6 months and the alterations in weight, soluble solid content, titratable acidity, firmness and microorganism development on fruit surface were examined. To better understand consumers’ attitude towards apple quality, the sensory analysis was performed. The results indicated that the most substantial positive effect on physical and chemical parameters and sensory quality has been achieved when ultra-low oxygen storage was applied. In general, all cultivars had retained better quality with regards to acids and soluble solids content. Likewise storage under ultra-low oxygen conditions, 1-Methylcyclopropene has positioned a strong positive effect on apple flesh firmness. Apples stored under different circumstances showed different contamination frequency and diversity of microorganisms, where the lowest incidence of microbial activity was found on apples stored under ultra-low oxygen, whereas the highest incidence of microbial activity was found on apples kept under storage in air.

KEYWORDS:

• Apple (Malus domestica)
• 1-MCP
• fruit contamination
• microorganisms
• ultra-low oxygen
• sensory evaluation

Introduction

Fresh fruits play a big role in our everyday diet because they are a basis of healthy diet due to biologically active substances—vitamins, minerals, organic acids and fibres that ensure protective properties against oxidative stress (Thilakarathna and Rupasinghe, 2013). Dietary fibre are one of the most useful parts of apple flesh and skin that work as brushes for our intestinal tract by removing useless substances from human organism, reduces the risk of gallstone diseases (Slavin and Lloyd, 2012). Apple is the most important fruit in Latvia. According to a research, conducted in 2011 by the Latvian Fruit-growers Association’s, 89% of the citizens preferred apples. About 50% of them selected fruit according to their taste and 39.6% according to their external quality. To preserve the fruit quality in terms of nutritional value and sensory indices during storage, appropriate technology should be chosen such as controlled atmosphere conditions. The right choice of gas composition in storage rooms as well as harvest fruits at optimal date (pre-climacteric stage) is the best way to save the storage potential of the fruits during long-term storage. At appropriate circumstances under ULO, storability of apples could be extended for more than a year, with an acceptable change in their sensory quality and almost no changes in their firmness (Ortiz et al., 2011; Varela et al., 2008). However, a previous study (Ortiz et al., 2010) showed that there is a major drawback of storage in CA—partial block of development of colour and aroma, which particularly impairs sensory qualities and has a negative impact on consumer choices. The degree of maturity is one of the most important factors determining fruit quality at-harvest and during long-term storage (Drudze, 2005, 2003). Early harvested apples have lack of nutrients, in particular, volatile compounds, which are responsible for aroma of fruit (Bangerth et al., 2012). Conversely, over-mature apples are susceptible to mechanical injury and several physiological disorders and they are very sensitive to low temperatures (Fonollosa et al., 2009; Skic et al., 2016). The composition of storage atmosphere has been suspected of being important and this calls for thorough elucidation (Thompson, 2015).

Decreasing oxygen content significantly suppresses the intensity of ethylene production and significantly reduces the firmness, loss of apple, resulting in better consumer acceptance after prolonged storage period in CA (Juhņeviča-Radenkova and Radenkovs, 2016). The favourable composition of gas for apple storage was chosen based on well-founded long-term research of Latvian scientist (Juhneviča et al., 2011), as well as, taking into consideration results found in other countries. This research was conducted with the aim of determining the optimal storage conditions which could ensure the sensory quality and reduce the intensity of microorganism proliferation during long-term storage of some apple cultivars grown in cool climate.

Material and methods

Research time and place

This study was conducted during two years (2010–2012) at the Processing and Biochemistry Department of the Institute of Horticulture, Latvia University of Agriculture in Dobele (latitude 56°36ʹ35.5″ N, longitude 23°17ʹ57.6″ E).

Data were recorded at the weather station in Dobele, Latvia (latitude 56°36ʹ35.0”N, longitude 23°17ʹ58.7”E) for the years 2010–2012.

Fruit

Apples of commercially available and widely grown cultivars (autumn cultivars: ‘Auksis’, ‘Orlik’, and winter cultivars: ‘Antej’, ‘Belorusskoje Malinovoje’, ‘Sinap Orlovskij’ and ‘Zarja Alatau’) were chosen for the experiments. All apple trees were grafted on the rootstock B9 and grown in the orchard according to integrated system at the same conditions. Plants were sprayed twice a year with 6.5 kg/ha⁻¹ CaCl₂. In 2010 trees were sprayed on 17th and 29th of July, while in 2011 on 13th of July and 1st of August. Ripening stage of the fruit were assessed by starch index using starch iodine (SI) test and Streifs’ index (Skic et al., 2016). SI was determined as follows: Ten apples were cut in half across the equator, submerged for one minute in lugol’s solution (30 g KI + 10 g I₂ in 1 L H₂O) and dried. The colour patterns that appeared after the iodine treatment were compared with colour reference charts, which rated SI using one (all stained) to ten (not stained) Eurofru scale (Ctifl, France) (Skic et al., 2016). Results were expressed as mean values of three independent ratings.

Streifs’ index was determined using the following equation:

where: F—firmness, kg cm⁻²; SSC—soluble solids content (TSS), °Brix; SI—starch index (on a scale from 1 to 10). Harvested fruits were matched with the requirements for long-term storage of apples grown in Latvia (Drudze, 2005, 2003).

Immediately after harvesting, apples were air-cooled for 24 h in a cooling chamber at 4°C ± 0.5°C. Forty fruits per cultivar/treatment/storage technology (ab. 6 kg) were sampled, placed in polypropylene boxes with perforated walls. The cooled down apples were divided into four groups for post-harvest storage: 1) cold storage: control, apples were stored in air—called “cold storage conditions—C”;

2) Cold storage + 1-MCP: treated apples with 1-MCP were stored in air—called “cold storage + 1-MCP treatment – 1-MCP”;

3) ULO1—2.00 kPa CO₂, 1.00 kPa O₂;

4) ULO2—2.50 kPa CO₂, 1.50 kPa O₂.

Storage in ULO was implemented in Fruit Control Equipment s.r.l., FC_E Industry, (Triuzli, Italy) for six months. All samples were stored at temperature +2°C ± 1°C and relative air humidity of 85%. The treatment with ethylene inhibitor 1-MCP (purchased from RandH: Rohm and Haas Company, Italy) was performed at 4°C ± 0.5°C in a gastight container for 24 h.

1-MCP powdery substance was dissolved in warm water +50°C ± 2°C by the ratio of 1:30, to the concentration 0.625 µl L⁻¹, according to Wawrzyńczak et al. (2007).

Sensory evaluation

The sensory attributes of apples were evaluated 15 well trained panellists (5 men and 10 women) aged between 25 and 50, using: Line scale evaluation, by the standard method ISO 4121:2003—Sensory analysis—Guidelines for the use of quantitative response scales. Each sample presented before was coded with randomly three digit numbers to reduce any possible bias. The panellists were provided with five slices of apples for every experimental sample and asked score them for different sensory attributes. To avoid unwanted browning, apples were cut just before being served and placed on each serving tray in a randomised order. To evaluate overall acceptability of apple (external quality) together with the slices served also whole uncut apple sample. To evaluate sensory attributes such as colour, aroma, taste, acidity, sweetness and juiciness for all apple samples, assessment was carried out using 12-point Line scale, where 12 cm—“like extremely”, 6—“neither like nor dislike”, 0 = “dislike extremely”.

Physicochemical analyses

All quality parameters were analysed at harvest and after storage. Ten apples were individually used for the analysis of flesh firmness (kg cm⁻²), soluble solids content (°Brix), total acids content (%). Flesh firmness (without peel) was measured on two opposite sides of each apple using digital penetrometer (model TR 53205, Italy), equipped with 11 mm diameter probe, peak destructive force was expressed in kg cm⁻².

Titratable acidity (TA) was determined using standard method (LVS EN 12147:2001) and quantified by titration of 1 ml of juice (automatic titration DL 21, Mettler Toledo, Swiss) with 0.1 M NaOH to a pH 8.1, expended amount of NaOH was expressed in percentage of malic acid, using equation: T.A. (as g/L malic acid) = mL NaOH × 0.67 g/L = ml NaOH × normality

Soluble solids content (SSC) determined using standard method (LVS EN 12143:2001). Ten apples of each cultivar were selected and ground with a hand blender Bamix® (Switzerland, model SwissLine, Liechtensteinn) into a puree and further, the content of soluble solids (in °Brix) was determined using a digital electronic refractometer (type Pal-1, Tokio, Japan).

Fresh weight loss determined with scaling method provided by Rab et al. (2012).

All analysis was made in triplicate.

Microbiological analysis

Microflora of apple surface was evaluated according to LVS ISO 18593:2007 standard microbiology of food and animal feeding stuffs—horizontal methods for sampling techniques from surfaces using contact plates and swabs. The microbiological analyses were carried out according to the following reference methods:

• for detection of mesophilic aerobic and anaerobic microorganisms were used standard LVS EN ISO 4833-2:2014 Microbiology of food chain—Horizontal method for the enumeration of microorganisms - Part 2: Colony count at 30 degrees C by the surface plating technique (ISO 4833-2:2013);

• Yeasts and moulds were determined according to standard LVS ISO 21527-1:2008 “Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of yeasts and moulds - Part 1: Colony count technique in products with water activity greater than 0.95;

• Pseudomonas spp. was determined according to standard LVS EN ISO 16266:2008 Water quality—Detection and enumeration of Pseudomonas aeruginosa—Method by membrane filtration;

• Microscopic fungi were identified according to the method provided by the group of scientists (Juhneviča et al., 2011).

Statistical analysis

Three replicates with ten fruits were used for each quality determination. Data analysis was carried out using the General Linear Model functions in the IBM® SPSS® Statistics programme 20.0 (SPSS Inc., Chicago, Illinois). The obtained data were analysed using descriptive statistics. Significant differences determined using UNIANOVA, by Least Significant Difference (LSD) criteria. The Analysis was done considering the main factor influence (storage conditions) on fruit quality. The significance of differences was determined at p < 0.05. Mean and standard deviation values were calculated for all parameters.

In order to compare sensory data that were obtained from Line scale evaluation, obtained results were processed by PanelCheck V1.4.2 programmed by Oliver Tomic and Henning Risvik software using Principal Component Analysis.

Results and discussion

Ripening stage of the fruits

To determine the optimal harvesting date (maturation) of any apple cultivar, many parameters must be determined before harvesting: flesh firmness, total soluble solids, acids content, ethylene concentration, sensory parameters and starch breakdown (Iodine-starch dates) (Skic et al., 2016). It is well-known that the optimal harvesting date differs for various cultivars depending on the results of iodine-starch test, for example, it is 5.0 for ‘Elstar’ and 8.0 for ‘Golden Delicious’ (Brookfield et al., 1997). A previous study indicated that the optimal Iodine-starch index for ‘Auksis’ is 5.0, for ‘Orlik’ is 4.0, for ‘Sinap Orlovskij’ is 4.0–4.8 and for ‘Zarja Alatau’ is 4.5–6.7 (Juhnevica-Radenkova et al., 2014).

Results in the current paper (Table 1) indicate that cultivars ‘Auskis’, ‘Orlik’, ‘Antej’, ‘Belorusskoje Malinovoje’ and ‘Sinap Orlovskij’ fruits harvested in 2010 had the most appropriate iodine starch indexes, while their harvesting dates were delayed in 2011 since their Streif index was lower (0.1). ‘Zarja Alatau’ apples had optimal index in both years (2010 and 2011).

Table 1. The parameters characterising the maturity stage of apples.

Cultivars	Indexes	The growing season
		2010/2011	2011/2012
‘Auksis’	Starch-Iodine	5^b	7^a
	Streif	0.2^a	0.1^b
‘Orlik’	Starch-Iodine	4^b	7^a
	Streif	0.3^a	0.1^b
‘Antej’	Starch-Iodine	4^b	6^a
	Streif	0.3^a	0.2^b
‘Zarja Alatau’	Starch-Iodine	5^b	6^a
	Streif	0.2^a	0.2^a
‘Belorusskoje Malinovoje’	Starch-Iodine	5^b	6^a
	Streif	0.2^a	0.2^a
‘Sinap Orlovskij’	Starch-Iodine	5^b	6^a
	Streif	0.2^a	0.2^a

Note: Mean value for the same cultivar and test followed by different small letters are significantly different by the LSD at 0.05 level

Changes of fruit quality during storage

Softening of apple during storage is a serious problem for many growers in Latvia. The biological causes of softening have been well studied in considerable researches, in order to manage this process effectively. As firmer apples tend to be juicier, crisper, crunchier and less mealy than soft fruit, rapid softening is generally considered as an undesirable ripening process in apple (Conforti and Totty, 2007). Results showed that after six months of storage (Table 2), average weight loss (for all cultivars and conditions) increased due to all storage circumstances and was significantly (p < 0.05) higher in those apples kept in air (average weight loss for all cultivars—10.71% (2010/2011) and 10.47% (2011/2012) in comparison to fruit kept in CA storage. The most pronounced shrinkage was detected for fruit those had very thin skin (‘Zarja Alatau’, ‘Аntej’, ‘Sinap Orlovskij’, ‘Belorusskoje Malinovoje’). Too high weight loss is explained by the long storage period which was 180 days long. Since most of the publications reflect only results, those obtained after 120 or 160 days of storage (Kårlund et al., 2014; Rab et al., 2012); therefore, a direct comparison of the current results is not possible.

Table 2. Fresh weigh loss of apples stored under different conditions, %.

Cultivars	Storage conditions	The growing season
		2010/2011	2011/2012
‘Auksis’	C	6.8^aB	7.8^aA
	1-MCP	6.7^aA	6.8^bA
	ULO1	5.1^bA	2.1^dB
	ULO2	4.1^cA	3.4^cB
‘Оrlik’	C	8.3^aA	7.4^aB
	1-MCP	6.6^cA	2.9^dB
	ULO1	7.3^bA	4.1^cB
	ULO2	6.9^bA	5.2^bB
‘Аntej’	C	10.7^aB	13.6^aA
	1-MCP	5.8^cA	5.9^cA
	ULO1	8.1^bA	7.3^bB
	ULO2	8.3^bA	7.6^bB
‘Zarja Alatau’	C	14.1^aA	13.5^aB
	1-MCP	7.5^bB	8.7^bA
	ULO1	6.8^cA	4.1^dB
	ULO2	7.1^bA	5.1^cB
‘Belorusskoje Malinovoje’	C	12.6^aA	10.4^aB
	1-MCP	9.7^bA	9.3^bA
	ULO1	4.2^dB	7.2^cA
	ULO2	5.2^cB	6.6^cA
‘Sinap Orlovskij’	C	11.8^aA	10.1^aB
	1-MCP	8.2^bB	9.8^aA
	ULO1	8.1^bA	7.3^bB
	ULO2	6.7^cA	5.4^cB

Note: Value for the same cultivar and year followed by different small letters are significantly different by the LSD at 0.05 level (differences between storage conditions). Value for the same cultivar and storage conditions followed by different capital letters are significantly different by the LSD at 0.05 level (differences between the years).

In turn, apples that treated with 1-MCP, had lost 7.42% and 7.23% of fresh weight in 2010/2011 and 2011/2012, respectively (Table 2). Apple fruit that were stored under ULO1 conditions showed lower weight loss 6.60% and 5.35% in 2010/2011 and 2011/2012, respectively. ULO2 conditions caused 6.38% and 5.55% weight loss in 2010/2011 and 2011/2012, respectively. It is evident that most fruits and vegetables become unmarketable when they lose 5%–10% of their fresh weight (Kasim and Kasim, 2012).

Two of the main reason for fresh weight loss is transpiration and respiration processes that take place throughout fruit storage. Considering the above-mentioned statement, seen that considerably positive results were achieved when 1-MCP treatment as well as controlled atmosphere storage technology is applied each alone, fresh weight losses were not exceeded the above-mentioned limit. Our findings are in agreement with the previously published paper provided by Juhņeviča-Radenkova and Radenkovs (2016) and Juhnevica-Radenkova et al. (2016), wherein mentioned that 1-MCP treatment significantly reduces fresh weight loss, delay of ripening-related biochemical and physiological changes, as well as decreases intensity of rotting. With regard to controlled atmosphere storage, similar observations had been found by the group of researchers (Palou et al., 2003), showing that apple storage under CA conditions ULO2 (2.50 kPa CO₂, 1.50 kPa O₂), can significantly reduce the fresh weight loss during long-term storage.

Apple flesh firmness

Apple flesh firmness is dependent on the degree of ripeness, place of growing, weather conditions and cultivar (Juhņeviča-Radenkova and Radenkovs, 2016). Flesh firmness decreases during fruit ripening; therefore for all cultivars the highest firmness has been observed before storage and storage technology significantly affects the firmness of the apples (Juhņeviča-Radenkova and Radenkovs, 2016).

Data that are depicted in Table 3 disclose that at the beginning of long-term storage, flesh firmness fluctuated in a range from 10.15 to 16.43 kg cm⁻² (2010/2011) and from 8.24 to 14.79 kg cm⁻² (2011/2012). As seen, there was a significant difference in firmness decline among all storage technologies during fruit storage, the changes were considerably slower in ULO1, ULO2 and cold storage +1-MCP than in cold storage. The percentage loss after six months of storage (2010/2011) for all apple cultivars was 33.83% in ULO1, 30.54% in ULO2, 36.68% 1-MCP treated, while apples those kept under cold storage lost an average value 39.53% of fresh weight. A more pronounced firmness loss was recorded during the second year (2011/2012). The results obtained are similar to those reported by Vanoli et al. (2009), who also found controlled atmosphere storage was able to maintain fruit firmness significantly better than conventional storage or cold storage, however the efficacy of controlled atmosphere storage in maintaining apple firmness might be year-dependent. However, better preservation of firmness loss has been obtained for samples 1-MCP treated (an average loss for all cultivars 59.47%) and for ULO1 (60.05%), while for cold storage (67.53%).

Table 3. Flesh firmness of apples stored under different conditions, kg cm⁻².

Storage conditions	‘Auksis’	‘Оrlik’	‘Аntej’	‘Zarja Alatau’	‘Belorusskoje Malinovoje’	‘Sinap Orlovskij’
Research year 2010/2011
Before storage	13.19^dA±0.52	15.87^bA±0.70	16.43^aA±0.60	14.33^cA±0.42	10.15^eA±0.57	10.85^fA±0.50
Cold storage	6.98^cD±0.35	7.18^cD±0.37	8.39^bD±0.36	10.45^aE±0.36	8.28^bC±0.24	6.41^dC±0.34
1-MCP	7.55^cC±0.38	6.78^dE±0.32	8.63^bCD±0.26	12.00^aC±0.47	8.37^bC±0.36	6.64^dC±0.38
ULO1	7.95^cB±0.45	8.10^cB±0.45	8.90^bC±0.38	11.18^aD±0.43	8.13^cC±0.41	7.96^cB±0.37
ULO2	8.22^dB±0.48	7.64^eC±0.26	9.33^bB±0.38	12.63^aB±0.41	8.80^cB±0.43	8.10^dB±0.43
Research year 2011/2012
Before storage	8.26^eA±0.00	8.24^eA±0.41	11.06^dA±0.00	13.39^bA±0.28	12.45^cA±0.48	14.79^aA±0.52
Cold storage	3.28^bcD±0.38	3.31^bcC±0.32	3.43^bC±0.00	4.21^aD±0.43	2.99^cD±0.00	4.21^aB±0.39
1-MCP	4.01^cB±0.43	4.66^bB±0.41	4.66^bB±0.26	5.23^aB±0.47	3.47^dC±0.36	4.29^cB±0.38
ULO1	3.70^cBC±0.42	4.41^bB±0.45	4.41^bB±0.45	5.38^aB±0.26	3.94^cB±0.56	4.39^bB±0.50
ULO2	3.56^bCD±0.31	4.56^aB±0.34	4.56^aB±0.30	4.71^aC±0.39	3.70^bBC±0.59	3.53^bC±0.45

Note: Mean value with standard deviation (±) for the same storage followed by different small letters are significantly different by the LSD at 0.05 level (differences between cultivars). Mean value with standard deviation (±) for the same cultivar followed by different capital letters are significantly different by the LSD at 0.05 level (differences between storage conditions).

Titratable acidity

The main acids in apples are: malic acid, citric acid and tartaric acid, and their contents depend on the cultivar and degree of ripeness (Cunha et al., 2002). During ripening content of acids in apples are reduced due to activity of endogenous enzymes (Fagundes et al., 2013) and oxygen exposure (Fonseca et al., 2002).

At harvest, the titratable acidity (TA) was statistically higher (p < 0.05) in year 2010/2011 than in 2011/2012 except cultivar ‘Auksis’ apples (Table 4). Moreover, the most pronounced TA was recorded in winter cultivar ‘Antej’, ‘Zarja Alatau’, ‘Belorusskoje Malinovoje’ and ‘Sinap Orlovskij’ apples. Differences in TA values between the years might be due to weather conditions, in particular temperature and lack of nutritive elements. After six month storage, a significant decline in TA was observed during all storage methods (Table 4). Deterioration of TA during storage was higher in season 2010 compared to season 2011. An average reduction for all cultivars was 41.03% (cold storage), followed by cold storage +1-MCP (37.77%), ULO2 (34.77%) and ULO1 (31.39) (Table 4). The same trend in decline of TA was observed within the second year (2011), where better preservation of organic acids was achieved when ULO storage was applied. Our results are similar to those provided by Weber et al. (2013), who pointed out that ULO storage promotes better preservation or organic matter due to suppresser respiratory activity. In terms of 1-MCP treatment, several researchers have reported that this technique allows considerably suppress the total acids loss during cold storage (Fan et al., 1999; Fan and Mattheis, 1999; Pre-Aymard et al., 2005; Saftner et al., 2003; Watkins et al., 2000; Zanella, 2003). However, our results indicated that treatment with 1-MCP had no positive effect on preservation of TA in apples, since efficiency of 1-MCP might be dependent on cultivar, ripening stage and year (Watkins, 2008).

Table 4. The changes in titratable acidity of apples while long-term storage, %.

Cultivars	Storage conditions	Growing season
		2010/2011	2011/2012
‘Auksis’	Before	0.51^aB ±0.01	0.65^aA±0.02
	C	0.43^aA±0.02	0.40^bA±0.01
	1-MCP	0.43^aA±0.03	0.50^bA±0.01
	ULO1	0.46^aA±0.04	0.44^bA±0.01
	ULO2	0.47^aA±0.04	0.40^bA±0.01
‘Оrlik’	Before	0.57^aA±0.01	0.41^aB±0.00
	C	0.33^bA±0.01	0.37^aA±0.02
	1-MCP	0.33^bA±0.02	0.38^aA±0.01
	ULO1	0.43^aA±0.02	0.39^aA±0.12
	ULO2	0.36^bA±0.02	0.41^aA±0.01
‘Аntej’	Before	1.20^aA±0.01	0.70^aB±0.02
	C	0.60^bA±0.01	0.57^aA±0.03
	1-MCP	0.63^bA±0.01	0.59^aA±0.04
	ULO1	0.57^bA±0.01	0.59^aA±0.01
	ULO2	0.66^bA±0.02	0.65^aA±0.02
‘Zarja Alatau’	Before	1.17^aA ±0.01	0.59^aB±0.00
	C	0.50^cA±0.01	0.49^aA±0.00
	1-MCP	0.60^cA±0.01	0.49^aA±0.01
	ULO1	0.83^bA±0.01	0.49^aB±0.01
	ULO2	0.66^cA±0.02	0.51^aA±0.01
‘Belorusskoje Malinovoje’	Before	1.05^aA±0.01	0.67^aB±0.00
	C	0.63^bA±0.02	0.34^bB±0.02
	1-MCP	0.66^bA±0.01	0.51^aA±0.01
	ULO1	0.66^bA±0.02	0.62^aA±0.01
	ULO2	0.66^bA±0.01	0.60^aA±0.02
‘Sinap Orlovskij’	Before	1.02^aA±0.01	0.82^aB±0.02
	C	0.60^bA±0.01	0.63^bA±0.02
	1-MCP	0.66^bA±0.01	0.65^bA±0.01
	ULO1	0.66^bA±0.01	0.67^bA±0.01
	ULO2	0.63^bA±0.01	0.66^bA±0.01

Note: Mean value with standard deviation (±) for the same cultivar and year followed by different small letters are significantly different by the LSD at 0.05 level (differences between storage conditions). Mean value with standard deviation (±) for the same cultivar and storage conditions followed by different capital letters are significantly different by the LSD at 0.05 level (differences between the years).

Total soluble solids content

Soluble solids content (SSC) in apples depends on cultivar, maturity stage, season, as well as weather conditions (Fonseca et al., 2008). Our results showed (Table 5) that SSC was not changed in many cases during storage. A significantly slower deterioration of SSC loss was found when apples were stored under controlled atmosphere conditions. However, in many cultivars of apples SSC was significantly increased. Considerable increase was noted for ‘Auksis’ (ULO1, ULO2 and 1-MCP, year 2010/2011), ‘Antej’ (ULO1 and ULO2, year 2011/2012), ‘Zarja Alatau’ (ULO2, year 2011/2012), ‘Belorusskoje Malinovoje’ (ULO2, year 2011/2012) and ‘Sinap Orlovskij’ ULO2, year 2011/2012), indicating perhaps a delayed respiration rate and ripening process. With regard to 1-MCP treatment, it is seen that in many cases there were no significant positive effect on preservation of soluble solids. In most cases, it decreased or remained intact. Similar observations were noted for those apples stored in cold storage. Based on literature a positive 1-MCP effect has not been observed on preservation of soluble solids content during pineapple (Selvarajah et al., 2001), papaya (Qiuping et al., 2006), apple (DeEll et al., 2002; Fan et al., 1999) and plum (Radenkovs et al., 2016) storage. However, the results from the current research reveal that strong positive effect on preservation of soluble solids can be achieved when a controlled atmosphere is applied. Our results are confirmed by the group of researchers from Poland (Wawrzyńczak et al., 2007), who stated that a significantly better preservation of soluble solids has been achieved using ULO storage.

Table 5. The changes in soluble solid content of apples while long-term storage, °Brix.

Cultivars	Storage conditions	Growing season
		2010/2011	2011/2012
	Before	11.71^bB±0.04	14.60^aA±0.04
	C	11.53^bB±0.04	13.22^bA±0.06
‘Auksis’	1-MCP	12.61^aB±0.04	13.56^bA±0.07
	ULO1	12.47^aB±0.03	13.39^bA±0.14
	ULO2	12.88^aB±0.05	13.30^bA±0.09
	Before	12.17^aA±0.05	11.52^aB±0.00
‘Оrlik’	C	12.28^aA±0.05	11.50^aB±0.07
	1-MCP	11.22^bA±0.05	11.62^aA±0.09
	ULO1	11.44^bA±0.10	11.57^aA±0.04
	ULO2	11.77^bA±0.02	11.62^aA±0.06
	Before	14.71^aA±0.02	11.29^bB±0.00
‘Аntej’	C	10.56^cA±0.06	10.54^cA±0.07
	1-MCP	10.70^cB±0.04	12.34^aA±0.01
	ULO1	11.17^bA±0.03	11.55^bA±0.16
	ULO2	10.59^cB±0.05	12.34^aA±0.05
	Before	13.51^aA±0.08	11.87^bB±0.05
‘Zarja Alatau’	C	12.55^bA±0.07	11.86^bB±0.02
	1-MCP	12.65^bA±0.07	11.94^bB±0.08
	ULO1	13.81^aA±0.09	11.93^bB±0.14
	ULO2	12.15^bA±0.18	12.65^aA±0.04
	Before	10.93^aA±0.14	10.25^bA±0.00
‘Belorusskoje Malinovoje’	C	10.58^aA±0.06	10.31^bA±0.00
	1-MCP	9.34^bB±0.02	10.42^bA±0.02
	ULO1	9.52^bB±0.05	10.76^bA±0.02
	ULO2	10.26^aB±0.07	11.55^aA±0.07
	Before	12.87^aA±0.04	11.84^bB±0.04
‘Sinap Orlovskij’	C	12.28^aA±0.05	11.81^bB±0.07
	1-MCP	12.18^aA±0.07	11.99^bA±0.02
	`ULO1	12.09^aA±0.04	12.10^bA±0.02
	ULO2	12.19^aB±0.02	12.76^aA±0.01

Note: Mean value with standard deviation (±) for the same cultivar and year followed by different small letters are significantly different by the LSD at 0.05 level (differences between storage conditions). Mean value with standard deviation (±) for the same cultivar and storage conditions followed by different capital letters are significantly different by the LSD at 0.05 level (differences between the years).

Apple sensory evaluation using line scale

Principal component PC analysis was performed on the sensory data of the six analysed apple cultivars before (Figure 1(a, b)) and after long-term storage (Figure 2(a, aa) and Figure 2(b, bb)). PC1 and PC2 together explain 74.5% (Figure 1(a) – growing year 2010/2011) and 93.4% (Figure 1(b) – growing year 2011/2012) of the samples’ variance, respectively. A clear separation among the samples is observed. Within the sensory evaluation cultivar ‘Auksis’ apples had been characterised as fruits with a pronounced taste and juiciness, while cultivar of ‘Zarja Alatau’ and ‘Belorusskoje Malinovoje’ apples were sourer than other samples. Distinctive aroma was inherent to cultivar ‘Orlik’ apple. The results from the second year of the research 2011/2012 indicate that a pronounced taste was inherent to cultivar ‘Auksis’ and ‘Antej’ apples. Besides, those apples had the most intense colour compared to other cultivars. According to panellists, cultivar of ‘Bellorusskoje Malinovoje’ apples was juicier among the other samples.

Figure 1. Biplot present scores and loadings of the first two principal components for apple sensory data before storage in 2010/2011 (a) and in 2011/2012 (b).

Note: Cultivars: AU—‘Auksis’, O—‘Orlik’, BM—‘Belorusskoje Malinovoje’, SO—‘Sinap Orlovskij’, ZA—‘Zarja Alatau’, A.—‘Antej’.

Figure 2. Biplot present scores and loadings of the first two principal components for apple sensory data after six months of storage in 2010/2011 (a—autumn cultivars, b—winter cultivars) and in 2011/2012 (aa—autumn cultivars, bb—winter cultivars).

Note: Letters represented in the figures indicate types of storage: C—cold storage, 1-MCP—cold storage + 1-MCP treatment, ULO1—controlled atmosphere conditions 1, ULO2—controlled atmosphere conditions 2; Cultivars: AU—‘Auksis’, O—‘Orlik’, BM—‘Belorusskoje Malinovoje’, SO—‘Sinap Orlovskij’, ZA—‘Zarja Alatau’, A.—‘Antej’.

As can be seen on score and loading plots, PC1 and PC2 together explain 83.1% (Figure 2(a)) of the autumn samples’ variance (growing year 2010/2011). A clear separation (Figure 2 top right square) has been obtained based on storage conditions. According to results from sensory evaluation, it is evident that after six months storage cultivar of ‘Orlik’ apples those treated with 1-MCP were sweeter and had pronounced colour, while cultivar of ‘Auksis’ apples were characterised as fruits with distinctive juiciness. In turn, ‘Orlik’ apple samples those stored under controlled atmosphere conditions in ULO1 and ULO2 were sourer in comparison to other samples. When analysing the results obtained from sensory evaluation of winter cultivars, it is seen that PC1 and PC2 together explain 66.2 (Figure 2(b)) of the samples’ variance. Panelists noted that sweetness and taste of ZA_ULO2, ZA_C, BM_C was the most intense (top left square), while SO_C apple samples that were kept under cold storage conditions were described as fruits with the most intense aroma. All of apple samples those stored under controlled atmosphere conditions as well as treated with 1-MCP in terms of colour had been characterised as “freshly picked from the tree”. However, three of apple samples had been characterised as sour with a distinctive juiciness: A_C, A_ULO2 and BM_ULO2.

Analysis of the research results obtained within the year 2011/2012 shows that PC1 and PC2 together explain 98.7% and 84.2% (Figure 2(aa) and Figure 2(bb)) of the samples’ variance.

Plots reveal that fruits that were stored under ULO1 and ULO2 conditions, had been described as acidic—‘Belorusskoje Malinovoje’ and ‘Antej’, respectively (Figure 2(bb)). In turn, panelists noted that fruit samples that were stored under ULO conditions, as well as treated with 1-MCP were characterised as fruits with pronounced taste ZA_ULO2 (2010/2011) and AU_1-MCP and AU_ULO2 (2011/2012). Panelists noted that the most intense sweetness was typical for those samples which were stored under normal atmosphere condition—ZA_C and BM_C, 1-MCP-treated—O_1-MCP and ULO2—ZA_ULO2 (2010/2011).

Results of identification of microorganisms dominating on the surface of apples

The results of research (Table 6) indicated that after cold storage, 92% of the total microflora on the fruit surface consisted of the following microscopic fungi: Penicillium rugulosum, Alternaria alternata (Figure 3(b)), Botrytis cinerea and Aspergillus terreus and bacteria: Bacillus cereus. A similar observation was found by other scientists (Juhneviča et al., 2011; Juhnevica-Radenkova et al., 2016). Spectrum of microorganisms isolated from apples which were treated with 1-MCP consisted of: Mucor circinelloides (Figure 3(a)), Candida sake, Pichia carsoni and Penicillium rugulosum. As can be seen, 31.70% of microflora on these apples was microscopic fungi and 21.79% were yeasts. After six months of apple storage under controlled atmosphere in ULO1, the following microorganisms were identified on the surface of fruits: Monilinia fructigena, Botrytis cinerea, Penicillium expansum, Fusarium avenaceum and Mucor circinelloides, while on the apples stored in ULO2 the following microorganism species and genera were identified: Monilinia fructigena, Fusarium avenaceum, Phomopsis/Diaporthe eres, and Neofabraea alba.

Table 6. A frequency of microorganisms isolated from apple fruit surface, %.

Type of storage	Identified microorganisms	Frequency of microorganisms isolatedfrom apple fruit surface, %
Cold storage	Aspergillus terreus	12.21
	Alternaria alternata	19.11
	Botrytis cinerea	14.15
	Penicillium rugulosum	35.16
	Bacillus cereus	12.11
1-MCP	Mucor circinelloides	16.66
	Candida sake	6.66
	Penicillium rugulosum	15.13
	Pichia carsoni	17.9
ULO1	Monilinia fructigena	15.69
	Botrytis cinerea	20.33
	Penicillium expansum	5.66
	Fusarium avenaceum	19.66
	Mucor circinelloides	5.78
ULO2	Monilinia fructigena	21.33
	Fusarium avenaceum	11.67
	Phomopsis/Diaporthe eres	11.67
	Neofabraea alba	23.33

Figure 3. Microflora of apples Mucor circinelloides. (a), Alternaria alternata. (b), after storage.

The results showed that after ULO1 and ULO2 storage, 76% and 68%, of all the microorganisms located on the surface of apples were microscopic fungi. The lowest prevalence of Penicillium spp., in ULO1 has been found compared with the fruits kept under cold storage. Our findings coincide with the results provided by a group of scientists (Juhnevica-Radenkova et al., 2016), stated that the genus of Penicillium has also been detected on apples that were stored under controlled atmosphere conditions, noted that Penicillium expansum and Penicillium roqueforti are able to well-grow in 2% of oxygen. Within the research, Fusarium avenaceum was found in both ULO conditions. Thus far, apple rotting caused by Fusarium avenaceum, have received a little attention, especially for those apples that were stored under ULO. According to the analysis of literature, only a few reports on Fusarium spp., were found. For instance, a group of researchers from Croatia (Sever et al., 2012) have found a relatively high incidence of Fusarium spp., of four apple cultivars. The frequency of Fusarium rot varied from 27.4% to 33.2%, and the most susceptible cultivar was Pink Lady. Authors also have declared that Fusarium rot may be a more important post-harvest disease in ULO storage conditions than expected; hence, Fussarium spp., should be evaluated in a more detailed analysis.

Currently, scientists pay more attention to those problems associated with the human health, and one of the issues for consideration “how to ensure global demand for qualitative and especially secure food products” (Juhnevica-Radenkova et al., 2016). For instance, some of the trials have been conducted using heat-treatment technologies that aimed to decrease Escherichia coli O157:H7 and Salmonella muenchen population, presented on the surface of apples (Wang et al., 2012). In addition, some of the evidence has been pointed out that hot water treatment reduces the development of such microscopic fungi: Botrytis cinerea, Penicillium spp., Mucor piriformis and Monilinia spp., (Mari et al., 2008; Sholberg and Randall, 2007). Our observations disclose that pathogenic microflora like Escherichia spp. and Salmonella spp., which harmful affecting human organism, was not detected.

Within the first year of research (2010/2011), in spite of the storage technology, obvious signs of bitter pit (Figure 4(a)) were found in cultivar ‘Sinap Orlovskij’ apples. While, in the year 2011/2012, when apples were kept under cold storage apparent signs of bitter rot (Figure 4(b)) on cultivar ‘Auksis’ apples were noted. Furthermore, fungal diseases such as blue mould (Figure 4(c)) and black rot with a high prevalence were also found.

Figure 4. Different disorders and diseases observed after storage, bitter pit (a), bitter rot (b), and diseases caused by microscopic fungi, blue mold (c).

Conclusions

The present study shows that after six months apple storage fresh weight loss was significantly higher in apples that were stored in cold storage compared to fruits stored under ULO or treated with 1-MCP. The most substantial positive effect on physical and chemical parameters and sensory quality has been achieved when Ultra-low oxygen (ULO) storage was applied. Likewise to storage under ULO conditions, 1-Methylcyclopropene (1-MCP) has positioned a strong positive effect on apple flesh firmness. In general, all cultivars had retained better quality in terms of acids and soluble solids content, when they were stored in ULO environment, while no preservation of organic compounds, such as titratable acidity and soluble solids was observed when 1-MCP technology was applied. Positive results have also been obtained from sensory evaluation of apple samples that were stored in ULO. Panellists noted that apples had a pronounced juiciness, colour, aroma, and acidity, while apples that before storage were treated with 1-MCP were sourer, juicier with a pronounced colour. Microbiological assessment of the surface of differently stored apple samples showed that frequency and diversity of microorganisms might be technology-dependent. The highest microbial diversity and amount on the apples were found in the cold storage, suggesting that these conditions were not suitable for long-term storage. The most prevalent microorganisms were microscopic fungi: Penicillium rugulosum, Alternaria alternata, Botrytis cinerea and Aspergillus terreus and bacteria: Bacillus cereus. Spectrum of microorganisms isolated from apple samples that before storage were 1-MCP-treated consisted of: Mucor circinelloides, Candida sake, Pichia carsoni and Penicillium rugulosum. Furthermore, incidence of proliferation was less severe. Storage of fruit in ULO appeared to be more promising technology for several commercially available cultivar of apples. Within the analysis performed did not found any bacterial contamination, besides, microflora of these apples consisted mainly from microscopic fungi—76% and 68%, respectively.

Additional information

Funding

This research has been prepared within the State Research Programme ‘Sustainable use of local resources (earth, food, and transport) – new products and technologies (NatRes)’ (2010.–2014.) Project no. 3. ‘Sustainable use of local agricultural resources for development of high nutritive value food products (Food) and The National Research Programme - Agricultural Resources for Sustainable Production of Qualitative and Healthy Foods in Latvia (AgroBioRes), project No. 10-4/VPP-7/3.