Evaluation of physico-chemical parameters of minimally processed apples packed in non-conventional modified atmosphere

Food Research International 37 (2004) 329–335 www.elsevier.com/locate/foodres

Evaluation of physico-chemical parameters of minimally processed apples packed in non-conventional modified atmosphere Pietro Rocculi *, Santina Romani, Marco Dalla Rosa Dipartimento di Scienze degli Alimenti, Facolta di Agraria, Universita degli Studi di Bologna, Sede di Cesena, Via Ravennate 1020, 47023 Cesena, Italy Received 14 April 2003; accepted 24 January 2004

Abstract Modified atmosphere packaging (MAP) with non-conventional gas mixtures in combination with non-sulphite dipping was tested during refrigerated storage on minimally processed (MP) apple slices for the physico-chemical characteristics. ‘Golden Delicious’ apple slices were dipped in an aqueous solution of 0.5% of ascorbic acid (AA), 0.5% of citric acid (CA), and 0.5% of calcium chloride (CC) to slow down the enzymatic browning. Apple slices were sealed in polypropylene boxes and conditioned in air (control) and in three different modified atmospheres (MAs) composed of 90% N2 , 5% O2 , 5% CO2 (A), 90% N2 O, 5% O2 , 5% CO2 (B) or 65% N2 O, 25% Ar, 5% O2 , 5% CO2 (C). The packed apple samples were stored at 4 °C for 12 days and pH, soluble solids content, weight loss, CO2 production, O2 consumption, texture changes, surface colour (whitening index, hue angle and chroma) and percentage of browning area were monitored. Beneficial effects of B and C MAP were found on enzymatic browning together with an increase of initial firmness and total soluble solid content. Atmospheres with high argon and nitrous oxide levels are shown some beneficial effect on the product quality during the 10 days period of storage, compared to control. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Apples; Minimally processed; Modified atmosphere packaging; Argon; Nitrous oxide; Shelf life

1. Introduction The major factors that affect fresh-cut fruit quality include cultivar (Kim, Smith, & Lee, 1993; Romig, 1995), pre-harvest growth and cultural practices (Romig, 1995), harvest maturity (Gorny, Hess-Pierce, & Kader, 1998), physiological status of the raw product (Brecht, 1995), post-harvest handling and storage (Watada, Ko, & Minott, 1996), processing technique (Bolin, Stafford, King, & Huxsoll, 1977; Saltveit, 1997; Wright & Kader, 1997), sanitation (Hurst, 1995), packaging (Cameron, Talasila, & Joles, 1995; Solomos, 1994) and temperature management during shipping, handling and marketing (Brecht, 1999). Factors controlling the shelf life of minimally processed (MP) fruits are a result of a complex process concerning a number *

Corresponding author. Tel.: +39-0547-636120; fax: +39-0547382348. E-mail address: [email protected] (P. Rocculi). 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.01.006

of physico-chemical and biochemical modifications that mainly affect flavour, colour and texture (Mencarelli & Massantini, 1994). Enzymatic browning is one of the principal quality deterioration factors of minimally processed apples. Browning reactions in apples become evident when, for instance, fruit is subjected to processing or mechanical injury (Laurila, Kervinen, & Ahvenainen, 1998). Extensive experiments have been performed to reduce undesirable browning and quality loss, by the application of antioxidant or reducing agents as the substitute for sulphites (US Patent, 5 939 117). Pizzocaro, Torreggiani, and Gilardi (1993) showed that different combinations of ascorbic acid (AA), citric acid (CA), and calcium chloride (CC) prevent enzymatic browning of sliced apples. According to Ponting and Joslyn (1972) sliced ‘Golden Delicious’ apples could be protected from browning by using a mixture of AA (0.5%) and CC (0.05%) at pH 7. Sapers and Douglas (1987) showed that the treatment on cut apple surface with 1% CA solutions

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containing 0.4–3.2% of AA is effective in the inhibition of browning. Modified atmosphere packaging (MAP) has become a widely used food preservation technique, which minimally affects fresh product characteristics, and receiving better consumer perception as a natural and additivefree techniques (Day, 1996). Most studies have been designed based on the effects of different CO2 and O2 levels on the fruit metabolism and to extend the shelf life of whole and MP vegetables (Beaudry, 2000; Gil, Gorny, & Kader, 1998; Lee, Haggar, Lee, & Yam, 1991; Mathooko, 1996; Watada et al., 1996). Recently, there has been a great interest in the potential benefits of using argon (Ar) and other noble gases for MAP applications (Mostardini & Piergiovanni, 2002; Spencer, 1995). Studies have shown that Ar can effectively inhibit the growth of certain micro-organisms, suppress enzymatic activities, and control degradative chemical reactions in selected perishable food products (Spencer, 1995), such as MP fruits (Powrie, Chiu, & Wu, 1990). Previous work in the field (Spencer, 1995) has demonstrated that noble gases are biochemically active, probably due to their enhanced solubility in water compared with nitrogen, and possible interference with enzymatic receptor sites, reducing the respiration rate. Moreover continuous nitrous oxide (N2 O) gas treatment has shown a significant ripening inhibition effect by extending the lag phase, which precedes the ethylene rise and delayed colour change in pre-climateric fruits of tomatoes and avocados (Gouble, Fath, & Soudain, 1995). Furthermore, Leshem and Wills (1998) have shown that N2 O inhibit ethylene action and synthesis in high plants. Argon and nitrous oxide are known to sensitise micro-organisms to other anti-microbial agents (Qadir & Hashinaga, 2001; Thom & Marquis, 1984) and are now permitted for food use in the EU, as miscellaneous additives (Day, 1996). Therefore, Ar and N2 O both have a direct effect on the extension the shelf life of fruits. To our knowledge a little data is available in literature regarding the influence of Ar and N2 O on the shelf life of MP fruits. This research is aimed at studying the use of MAP with non-conventional gas mixtures (Ar and N2 O) in combination with non-sulphite dipping, for the inhibition of enzymatic browning and for some qualitative characteristics maintenance of MP apple slices, during refrigerated storage.

2. Materials and methods ‘Golden Delicious’ (Malus sylvestris L.) apples were harvested in November in Valtellina (Italy) and stored for 2 months at 2 °C in controlled atmosphere (1% O2

and 2% CO2 ). Subsequently, the fruits were purchased from a local market at commercial maturity. The ‘Golden Delicious’ variety was chosen, after the preliminary trials of different apple varieties on the bases of its less susceptibility to browning. After washing in tap water the apples were hand-peeled, cored and cut, using a sharp knife into slices of 1 cm thickness. For polyphenoloxidase (PPO) inhibition and maintenance of texture, apple slices were immediately dipped in aqueous solution of 0.5% of citric acid (CA), 0.5% of ascorbic acid (AA) and 0.5% of calcium chloride (CC) at 25 °C for 10 min. All reagents used were of analytical grade (Sigma Chemical, Co.). This kind of dipping treatment was chosen because after a comparison with other kinds of similar dipping treatment in preliminary trials, it showed to preserve apples better from browning. Preliminary trials were carried out using AA, CA and CC, in different proportion, for 5–15 min treatment time. After gently drying with blotting paper, 200 g of apple slices (about 15 slices) was put into polypropylene boxes and wrapped with polypropylene film (200 lm thick). Boxes and films were purchased by Hot Mould System (Torino, Italy). The permeability data of the plastic film at 25 °C were the following: water vapour transmission rate (WVTR): 0.3–0.6 (g/m2 /24 h) at 60% RH; O2 TR: 50–70 (cm3 /m2 / 24 h); CO2 TR: 15–25 (cm3 /m2 /24 h). The samples were packed using a quaternary gas mixer (CVC, Milano, Italy) and a compensated vacuumpacking welding machine (Food Basic, Vigevano, Italy) with the following atmospheres: Air (A); 90% Nitrogen (N2 ), 5% Carbon dioxide (CO2 ), 5% Oxygen (O2 ) (B); 90% Nitrous oxide (N2 O), 5% CO2 , 5% O2 (C); 65% N2 O, 25% Argon (Ar), 5% CO2 , 5% O2 (D). The samples were stored in a refrigerator at 4 (
1) °C for 12 days and analysed after dipping just before pack (zero time) and on the 4th, 8th and 12th day of storage. Fifteen samples for each packaging condition were prepared and five samples were randomly taken at each storage time (4, 8 and 12 days).

2.1. Physico-chemical determinations Total soluble solids (°Brix) content was determined at 20 °C by refractometry using a digital refractometer (PR1, Atago, Japan) on the juice obtained from apple slices, after filtering through Whatman #1 filter paper. pH was determined using an electronic pH meter (AMEL 334-B, Milano, Italy). Soluble solids and pH were carried out for each sample on the juice obtained by 10 apple slices, taken from the five replicates of each sample (two slices per box).

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Weight loss percentage was estimated after each period of storage by weighing all sample boxes. Texture analysis was performed at room temperature (20
2 °C), about 1 h after removing samples from 4 °C, by measuring the energy necessary for penetration of a 6 mm diameter stainless steel cylinder for 6 mm in apple slice tissues, using a texture analyser mod. HD500 (Stable Micro Systems, Surrey, UK) equipped with a 50 kg load cell. Test speed was performed at 0.5 mm per second and data were expressed as N  s. At each storage time, 10 measurements were obtained for each packaging condition from the five replicates, with two measurements for each sample. Average data for each sample was obtained from penetration tests carried out on 10 slices. CO2 –O2 levels measurements inside all samples boxes during storage were performed with a check point O2 / CO2 (PBI Dansensor, Milano, Italy). The apparatus is based on an electrochemical sensor, which is able to record O2 content and a mini-IR spectrophotometer able to record CO2 in the package atmosphere (accuracy: 0.1% O2 ; 2% CO2 ). The instrument was calibrated with O2 and CO2 air percentages. Parameters are expressed as DCO2 and DO2 percentage referred to initial values. 2.2. Colour measurements 2.2.1. Tristimulus reflectance colorimeter Surface colour was measured using a reflectance colorimeter (Chromameter-2 Reflectance, Minolta, Japan), equipped with a CR-300 measuring head. Colour was measured using the CIE L , a , b scale. Illuminant ‘‘C’’ (6774 K) was used. The instrument was calibrated with a white tile (L ¼ 98:03, a ¼ 0:23, b ¼ 2:05) before the measurements. Numerical values of L , a and b were converted into whiteness index: WI ¼ 100  ðð100  L Þ2 þ a2 þ b2 Þ1=2 (Bolin & Huxsoll, 1991); hue angle: h° ¼ tan1 ðb =a Þ and chroma: C  ¼ ða2 þ b2 Þ1=2 (McGuire, 1992). At each storage time, 10 readings were obtained for each packaging condition from the five replicates with two measurements for each replicate. 2.2.2. Image analysis The tristimulus approach to colour measurements was compared with an image analysis that was used to evaluate the colour because the surface of MP fruits does not usually homogeneously become brownish. The use of image analysis permits colour evaluation to all product areas at the same time (Russ, 1995). Images of MP apples were obtained by digitalisation using a colour plane scanner (Scan Jet 6300 C) (Fig. 1(a)). All images were obtained at the same conditions (true colour – 24 bit, resolution of 300 bit  pixel) by positioning on the scanner the apple slices of each sample,

Fig. 1. Apple slice images obtained by digitalisation using a colour plane scanner (a), isolated from the background (b) and virtually coloured on the bases of different levels of browning areas, calculated with advance Image Analysis Software (c).

held in a black box, in order to exclude the surrounding light. After acquisition, digitalized apple slice images were isolated from the background using the software PhotoshopÒ v. 5.0 (Adobe Systems Incorporated, USA) (Fig. 1(b)). With advance Image Analysis Software (Image ProPlusÒ v. 4.1, Media Cybernetics, USA), using RGB scale, apple images were evaluated in two steps: selection of total sample area and of different levels browning areas. On the bases of chromatic characteristics of all samples a colour model was built (Reale, Coppola, Cipriano, D’Acierno, Sorrentino, & Addeo, 2003; Rocculi, Romani, Tonizzo, & Dalla Rosa, 2003). The same colour model was applied to all apple slice images. Browning areas are selected in pixel within the images. The software, examining all pixels in the image, calculates browning area percentage in total (Fig. 1(c)). The measurements were obtained from the same slices used for colour determinations made with colorimeter.

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2.2.3. Statistical analyses Analysis of variance (ANOVA) and the test of mean comparison according to Fisher least significant difference (LSD) were applied; level of significance was 0.05. The statistical package STSG Statistica for Windows, version 6.0 (Statsoft Inc., Tulsa, UK) was used.

3. Results and discussion Data on the effects of different adopted atmospheres on some physico-chemical characteristics of the apple packed with different methods, investigated during refrigerated storage, are shown in Table 1. pH values and soluble solids content did not substantially change during the storage period for samples packed with A and B methods, while there was a slight increase of pH in C packaging method and of pH and soluble solids in apple slices packed with D method as they underwent ripening. Weight loss (%) was not remarkable and it did not change very much in any of the samples that probably had not lost water due to transpiration and respiration, as seen on a visual examination of the inside of the pack. Firmness values increased in all MP apples packed in the different methods, much more in C and D after 12 days of storage. The general firmness increase may be explained by the effect of CaCl2 used in dipping solutions; this result is confirmed in other experiments, where infiltration of calcium into fresh apple tissue and processed apples has been used to maintain firmness (Abbott, Conway, & Sams, 1989; Glenn & Poovaiah, 1990; Johnson, 1979; Mason, 1976; Sams & Conway,

1984). However, the greatest firmness increase, at the acceptable levels, of apples in C and D packs could probably be due to the effect of the non-conventional modified atmospheres. As far as the changes in percentage of CO2 inside the bags are concerned, apples in packs C and D showed the lowest production during storage time. The MP apples in air (A) showed the highest CO2 production and O2 consumption as it underwent the greatest metabolic changes although pH and soluble solid contents did not change very much. After 8 days of storage, the oxygen level inside sample boxes packed with B, C and D methods was zero. The changes of the physico-chemical characteristics of MP apple slices over time are probably due to the effect of adopted package methods; this because, on the basis of preliminary studies carried out on apple slices packed in the same MAP conditions, the total microbial and yeast load values remained under 103 CFU/g fresh weight. To compare the effect of different adopted atmospheres on MP apples, the ANOVA analysis was performed on the physico-chemical parameters during storage time (Table 2). Apples packed with A and B methods showed significantly lower pH values than C and D samples. In terms of soluble solids content, samples A and D showed significant differences from B and C. Only MP apples in A showed a significant difference in weight loss compared to the others. Sample D significantly differed from A as far as the firmness was concerned. Moreover, the apples packed in air (A) underwent the greatest and significantly different

Table 1 Physico-chemical characteristics of apple slices packed with different methods (A: air; B: 90% N2 , 5% CO2 , 5% O2 ; C: 90% N2 O, 5% CO2 , 5% O2 ; D: 65% N2 O, 25% Ar, 5% CO2 , 5% O2 ) during storage time at 4 °C Packaging method

Time (d)

A

B

C

D

a

Physico-chemical characteristicsa pH

DCO2 (%)

DO2 (%)

Soluble solids (°Brix)

Weight loss (%)

Firmness (N  s)

0 4 8 12

3.77
0.01 3.76
0.01 3.71
0.00 3.78
0.00

– 11.05
0.07 18.95
0.07 28.35
0.07

– )11.30
0.14 )20.70
0.14 )20.80
0.00

10.40
0.00 10.90
0.10 10.80
0.10 10.89
0.01

– )0.17
0.00 )0.08
0.00 )0.03
0.00

52.40
0.14 55.60
0.14 56.95
0.07 57.02
0.67

0 4 8 12

3.77
0.01 3.67
0.00 3.81
0.02 3.85
0.01

– 11.30
0.14 20.50
0.14 27.00
0.14

–

10.40
0.00 10.33
0.12 10.03
0.06 10.39
0.01

–

0.05
0.21 )5.00
0.00 )5.00
0.00

0.29
0.02 0.35
0.00 0.24
0.00

52.40
0.14 57.20
0.28 62.50
0.28 61.60
2.40

0 4 8 12

3.77
0.01 3.95
0.03 4.07
0.03 4.05
0.02

– 14.10
0.42 23.70
0.28 24.80
0.28

– )4.85
0.21 )5.00
0.00 )4.95
0.07

10.40
0.00 9.97
0.12 10.54
0.00 9.34
0.00

– 0.21
0.14 0.30
0.08 0.14
0.08

52.40
0.14 53.50
0.71 62.63
0.52 68.30
0.44

0 4 8 12

3.77
0.01 4.00
0.03 4.02
0.02 4.02
0.08

– 14.15
0.07 23.10
0.42 20.10
0.00

– )4.95
0.07 )5.00
0.00 )4.90
0.00

10.40
0.00 11.93
0.06 11.93
0.06 10.53
0.06

– 0.33
0.01 0.36
0.01 0.34
0.01

52.40
0.14 56.25
0.35 67.73
0.04 71.15
0.21

Average and
SD.

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Table 2 ANOVA analysis results between physico-chemical data of apple slices packed with different methods (A: air; B: 90% N2 , 5% CO2 , 5% O2 ; C: 90% N2 O, 5% CO2 , 5% O2 ; D: 65% N2 O, 25% Ar, 5% CO2 , 5% O2 ) Packaging method A B C D MSE d.f. n a–c A

Physico-chemical characteristicsA pH

D CO2 (%) b

a

3.75 3.77b 3.96a 3.96a

14.59 14.70a 15.65a 14.34a

0.009 76 80

102.51 76 80

D O2 (%)

Soluble solids (°Brix)

Weight loss (%)

Firmness (N  s)

)13.20 )2.45a )3.70a )3.71a

b

10.75 10.29c 10.07c 11.45a

b

)0.07 0.22a 0.16a 0.26a

55.49b 58.42ab 59.21ab 61.88a

23.28 76 80

0.219 76 80

0.016 76 80

32.684 156 160

b

Values in the same column followed by different letters differ significantly at p < 0:05 level. Average values of each sample calculated during the whole experiment (12 days).

Table 3 ANOVA analysis results between colour data of apple slices packed with different methods (A: air; B: 90% N2 , 5% CO2 , 5% O2 ; C: 90% N2 O, 5% CO2 , 5% O2 ; D: 65% N2 O, 25% Ar, 5% CO2 , 5% O2 ) Packaging method A B C D MSE d.f. n a–c A

Whitening indexA

Hue angleA

70.17a 69.00a 72.71b 75.14c

101.15a 101.02a 103.97b 103.63b

7.247 156 160

2.210 156 160

ChromaA 21.96c 21.77b;c 20.68a;b 20.35a 6.137 156 160

Browning area (%)A 37.06a 37.37a 20.73b 10.11b 385.25 44 48

Values in the same column followed by different letters differ significantly at p < 0:05 level. Average values of each sample calculated during the whole experiment (12 days).

consumption of O2 , compared to all MA packed samples. In general, on the basis of these results, it is possible to assess that the MP apples that had shown greater differences from each other were those packed with A and D methods. The changes in colour measurements seem to be able to discriminate the samples better than the other quality characteristics considered. On the basis of ANOVA results (Table 3) and as shown in Figs. 2–5, apple slices packed with A and B methods differ significantly from C and D, in particular in terms of whitening index (WI) and hue angle (h°) values. Treatment C did not show grate changes in WI and h° parameters during all storage time (Figs. 2 and 3). Treatment D however increased WI value and brightened after 8 days of storage. Apple slices packed with A and B methods had lower WI and h° values just at the beginning of the storage period, becoming darker than the others. Moreover, the same samples had a more saturated colour (Fig. 4). MP apples packed in modified atmospheres C and D, with alternative gas mixtures, seem to preserve their original colour better. These results have been confirmed by those obtained from image analysis (Table 3 and Fig. 5). In fact, apple slices in A and B packs showed the highest percentage (about more than 40%) of browning area, in particular

after the 4th day of storage. MA apples in pack D (65% N2 O, 25% Ar, 5% CO2 , 5% O2 ) showed the lowest browning level (15% of browning area) after 12 days of storage. This could be due to higher solubility of Ar than of N2 and to its competition with O2 at chemical-enzymatic level; in fact argon having the same solubility and

Fig. 2. Whiteness index values of MP apple samples during refrigerated storage. Packaging methods: A (jÞ: air; B (N): 90% N2 , 5% CO2 , 5% O2 ; C (dÞ: 90% N2 O, 5% CO2 , 5% O2 ; D (s): 65% N2 O, 25% Ar, 5% CO2 , 5% O2 .

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Fig. 3. Hue angle values of MP apple samples during refrigerated storage. Packaging methods: A (jÞ: air; B (N): 90% N2 , 5% CO2 , 5% O2 ; C (dÞ: 90% N2 O, 5% CO2 , 5% O2 ; D (s): 65% N2 O, 25% Ar, 5% CO2 , 5% O2 .

Fig. 4. Chroma values of MP apple samples during refrigerated storage. Packaging methods: A (jÞ: air; B (N): 90% N2 , 5% CO2 , 5% O2 ; C (dÞ: 90% N2 O, 5% CO2 , 5% O2 ; D (s): 65% N2 O, 25% Ar, 5% CO2 , 5% O2 .

molecular weight as O2 causes PPO inhibition, thus replacing it (Day, 1996). In our experiment, these effects could be enhanced due to the low packaging permeability that permitted retention of MA inside the boxes and its interaction with the product. Therefore, image analysis could be considered as a useful tool for defining or following the quality characteristics, in terms of colour and metabolic activities of this kind of product.

Fig. 5. Percentage of browning areas of MP apple samples during refrigerated storage. Packaging methods: A (jÞ: air; B (N): 90% N2 , 5% CO2 , 5% O2 ; C (dÞ: 90% N2 O, 5% CO2 , 5% O2 ; D (s): 65% N2 O, 25% Ar, 5% CO2 , 5% O2 .

ment could permit the fresh quality maintenance of MP apples for 12 days. In particular, modified atmosphere with argon, nitrous oxide, with a low percentage of CO2 and O2 (D) resulted the best mixture of tested gases in terms of PPO inhibition and lowering metabolic activities. Changes of some properties, such as pH, soluble solids and weight loss, did not well differentiate the samples, showing a behaviour not always significant and clear; on the contrary the firmness increased for all samples, remaining in all cases at acceptable levels. The changes in colour measurements and in the percentages of respiration gases inside packaging (in particular CO2 ) seem to distinguish the different packed MP apples better. Further studies are necessary to determine the sensory profile and the microbiological stability throughout the potential 12 day shelf-life, obtained when combining dipping treatment and non-conventional atmosphere (with Ar and N2 O) on MP apple slices.

Acknowledgements The authors acknowledge the technical support and gas supply provided by SAPIO srl and Dr. Alessandro Bacci for his excellent technical assistance.

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