based edible coating in minimally processed pumpkin

International Journal of Food Science and Technology 2018

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Original article Vacuum impregnation of chitosan-based edible coating in minimally processed pumpkin Ariana de Souza Soares,1* Afonso Mota Ramos,1 Erica Nascif Rufino Vieira,1 Ellen Silva Lago Vanzela,2 1 Patrıcia Martins de Oliveira & Daniele de Almeida Paula1 1 Food Technology Department, Federal University of Vicßosa (UFV), P.H. Rolfs Avenue, Campus, 36570-900 Vicßosa MG, Brazil 2 Department of Food Engineering and Technology, Institute of Biosciences, Letters and Exact Sciences, Campus of S~ao Jose do Rio Preto, 15 R. Crist ov~ ao Colombo, 2265, Jardim Nazareth, S~ao Jose do Rio Preto, SP, 16 15054-000, Brazil (Received 24 January 2018; Accepted in revised form 9 April 2018)

Summary

The goal of this research was to evaluate the use of vacuum impregnation (VI) and soaking techniques (ST) in the application of edible coatings of chitosan and chitosan + lauric acid to minimally processed pumpkins (MPP). The vacuum impregnation method led to greater component incorporation (5.9% and 1.75%, respectively) in the pumpkins when compared to soaking and consequently the formation of more uniform, thicker coatings (25.6 and 22.3 lm, respectively). However, VI caused greater changes in pH, acidity, colour and firmness. Relating to water content and carotenoid content, noncoated pumpkins presented greater losses during the storage period, regardless of impregnation method. The pumpkins with edible coatings, regardless of method, presented lower numbers of psychrotrophic micro-organisms and coliforms during the storage period. Therefore, soaking was considered the best method for the application of chitosan-based edible coatings to minimally processed pumpkins, as it led to smaller changes in the properties of the product.

Keywords

Edible coating, minimally processed, pumpkin, soaking, vacuum impregnation.

Introduction

Pumpkins are used for sustenance in humans and animals, and they encompass a great number of species from the Cucubitaceae family, many with economic importance in world horticulture (Cortez-Vega et al., 2014). It is a large vegetable, which brings challenges to its commercialisation, storage and handling, leading to product loss. On account of that, it is a vegetable with the potential to expand in the market of minimally processed foodstuffs, increasing its sales and adding to its value (Santos et al., 2016). In carrying out steps to acquiring minimally processed products, damage derived from cutting and slicing hastens the physiological responses of the vegetable, promoting reactions that diminish shelf life and favour microbial growth (Rico et al., 2007; Ranjitha et al., 2017). The application of edible coatings can be an alternative to prolong shelf life by promoting a barrier against gases, humidity and microorganisms. (Lin & Zhao, 2007; Leceta et al.,2015). *Correspondent: Email: [email protected]

doi:10.1111/ijfs.13811 © 2018 Institute of Food Science and Technology

Among the biopolymer-based coatings, chitosan is one of the most promising ones (Leceta et al., 2015). It is a cationic polymer, nontoxic, biodegradable, of high molar mass, with antimicrobial activity and the capacity to form films. (Zheng & Zhu, 2003). The film derived from chitosan acts as an excellent barrier to gases, although it is highly permeable to water vapour. The latter property can be minimised by a combination with other hydrocolloids or lipid compounds (Elsabee & Abdou, 2013). Application of coating can be carried out by different methods, soaking being the most common. However, depending on the viscosity of the solution and surface extensibility of the sample, the level of component retention in the produce to form a film is low. Thus, vacuum impregnation (VI) emerges as an alternative to enhance retention, forming thicker coatings, with higher uniformity and adherence (Vargas et al., 2009). This method consists in the exchange of gas or liquid imprisoned in the pores by an external liquid, through the hydrodynamic mechanism (HDM) prompted by pressure changes (Fito, 1994; Fito et al., 2001).

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VI and ST for addition of edible coatings in MPP A. S. Soares et al.

Vacuum impregnation is commonly used to introduce dissolved or dispersed substances, such as calcium salts, probiotics, antioxidants, among others, in the porous matrix of foodstuffs. (Occhino et al., 2011; R€ oßle et al., 2011; Oliveira et al., 2017). Vargas et al. (2009) researched the use of VI in the application of edible coatings to carrots; however, literature still lacks studies on the application of edible coatings to minimally processed pumpkins through VI. In this study, coatings of chitosan and chitosan plus lauric acid were applied to minimally processed pumpkins (MPPs) by vacuum impregnation and soaking. The goal was to evaluate and compare different application techniques and coating composition on product properties with the intention to prolong shelf life. Methods

Layout of experiment

The experiment was carried out in two steps. The first consisted in determining the optimal duration of vacuum application, to be used when applying chitosan-based coatings by vacuum impregnation at 500 mmHg. The durations tested were 2, 4, 6 and 8 min. Tests were carried out to identify pressure levels that caused minimal alteration to produce composition and its physical properties, and a working pressure of 500 mmHg was stipulated (data not shown). The second step consisted in comparison between the methods of vacuum impregnation (500 mmHg) and soaking, regarding application of coatings (chitosan and chitosan + lauric acid) in minimally processed pumpkins, employing the optimal time from the first step. Preparation and characterisation of coating-forming solution

Chitosan (1.5% w/v) (Sigma-Aldrich) was dispersed in aqueous solution of acetic acid (1% v/v) remaining under agitation for 16 h at room temperature (23 °C). Shortly after, Tween 40 (0.1% v/v) was added and the solution remained another 30 min under agitation. Afterwards, the solution was vacuum filtered by use of a Buchner funnel and a Kitassato, which was attached to a vacuum pump. Filter paper with 28-lm pores was used in filtration. Lauric acid (2% w/v) was added to half of the solution, and the mixture (chitosan + lauric acid) was emulsified at room temperature using Ultra turrax IKa 25 at 2268 g for 4 min (Wong et al., 1992; Assis & Alves, 2002; Vargas et al., 2009). The rheological behaviour of the coating-forming solutions was determined in a Brookfield rotational rheometer, model R/S plus SST 2000, programmable, with its interface attached to a microcomputer with


RHEOCALC V 1.1 software. A CC45 sensor (concentric cylinders) with strain rate varying from 0 to 300 s1 was used, for 2 min for increasing rotation rate and another 2 min for decreasing rotation rate, with measurements every 4 s. The superficial tension was determined by pendant drop method in Goniometer (KrussÒ, Germany), selecting the Young–Laplace fit as the calculation method. Shortly after film-forming solutions were prepared, analyses were carried out at 25 °C. Minimal processing of pumpkin

Pumpkins of the Japanese variety (Cucurbita maxima Duchesne x Cucurbita moschata Duchesne) were acquired from the food trade of Vicßosa, Minas Gerais, Brazil. They were diced into 2 9 2 cm cubes, sanitised in a chlorinated solution of 200 mg L1 of active chlorine for 10 min at 5 °C, rinsed in a chlorinated solution of 30 mg L1 for 5 min at 5 °C and centrifuged to remove excess water. Determination of duration of vacuum impregnation

Minimally processed pumpkins were immersed in filmforming solutions of chitosan and underwent 500 mmHg vacuum pressure for 2, 4, 6 and 8 min. Next, atmospheric pressure was re-established for the same period of time as vacuum pressure was applied in the previous step. Afterwards, pumpkins were placed in the packages of polyethylene terephthalate (PET) and stored at 5 °C for 16 days. On day 0 of storage, firmness, water content and coating thickness were measured, and on day 16 of storage, water content was measured. Analyses were performed in triplicate, and the experiment was carried out in three repetitions. Coating application by VI and soaking

After minimal processing, pumpkins were immersed in film-forming solutions of chitosan and chitosan + lauric acid for 8 min at atmospheric pressure. Regarding VI, the solutions containing immersed vegetables were put under 500 mmHg vacuum pressure for 4 min, followed by a period where atmospheric pressure was restored for the same amount of time, as previously defined. Control samples underwent the same treatment, but were submerged in aqueous solution of acetic acid (1% v/v) with pH adjusted to 4,0 by the use of NaOH 1 N. After application, regardless of method, pumpkins were drained in sieves and arranged in stainless steel plates, where they underwent drying process by forced air circulation, at 25 °C per 1 h. Then, minimally processed pumpkins were placed in polyethylene terephthalate (PET) packages and stored

© 2018 Institute of Food Science and Technology


at 5 °C for 16 days. Analyses were done on days 0, 4, 8, 12 and 16 after processing. All analyses were performed in triplicate, and the experiment was carried out in three repetitions. Chemical and physical characteristics Titratable acidity, potential of hydrogen (pH) and soluble solids content (SSC)

Acidity was expressed by citric acid percentage and measured by titration with 0.1 N of NaOH solution and using phenolphthalein as the indicator. The pH analysis was performed by direct reading of a Tecnopon potentiometer. Soluble solids content was measured using ABBE refractometer (model 100 RTA) (AOAC, 2000).

mi: MPP weight, impregnation method m: MPP weight, impregnation method

after undergoing soaking or (grams); before undergoing soaking or (grams).

Firmness

MPP firmness was measured in TA-TX texturometer (Texture Technologies Corp./Stable Microsystems), with a 25 mm diameter cylinder probe (Aluminum Cylinder Probe SMS, P/25). Samples were compressed to 30% of their original height in a compression cycle of 1 mm s1 velocity and a load cell of 0.05 N. Firmness indexes were calculated from characteristic curves of texture profile generated in Texture Expert Stable Micro Systems software. Coating thickness

Water content

Water content was measured by the gravimetric method, based on weight loss of samples that underwent heating at 105 °C in oven, as outlined in the Analytical Standards of the Adolfo Lutz Institute (2004). Total carotenoids

Carotenoids content was determined by spectrophotometric analysis, as outlined by Rodriguez-Amaya (2001). Acetone was used as the extractor solvent, and total carotenoids were measured at 450 nm. Results were expressed as mg of total carotenoids per g of pumpkin. Colour

Color Reader CR-10 (Minolta) was used to evaluate superficial colour of all treatments. Colour determination was conducted by direct reading of coordinates L*, a* and b* reflectance using the CIELAB L* scale, with three readings performed for each sample in different parts of the product in order to obtain an average. In addition to base coordinates, for total colour variation (DE) the following equation was used: ΔE = [(ΔL*)² + (Δa*)² + (Δb*)²]1/2 (Adekunt et al., 2010). Evaluation of component incorporation after application of coating

Incorporation was determined immediately after processing by weighing in an analytical balance, setting a relation between the weight of the minimally processed pumpkins before and after coating application. Results were expressed in percentages and were calculated from the following equation: IC ¼

ðmi mÞ 100 m

In which: IC: incorporation index;


Thickness was measured according to Botrel et al. (2007) with adjustments. After application of coating, previously coloured with methylene blue, cross sections were made of each sample for thickness measurement. Photographs were taken with an optical microscope at 49 magnification. ImageJ software provided coating thickness measurements. Scanning electron microscopy (SEM)

Analysis was carried out according to Martins et al. (2016), with the goal of verifying the microstructure of coating surface. To enable viewing of coating, the external part of the pumpkin was turned in the direction of the electron beam. Microbiological analyses

Psychrotrophic organisms were counted according to Cousin et al. (2001) using plate count agar (PCA) and incubated at 7 °C for ten days. Coliforms were counted at 30 °C and 45 °C through the most probable number method (MPN), according to Kornacki & Johnson (2001). Results were expressed as MPN per gram of pumpkin. Results and discussion

Characterisation of coating-forming solution

Coating-forming solutions were characterised in terms of surface tension and apparent viscosity (ẙ = 100 s1) at 25 °C. Surface tension of coating-forming solutions differed significantly (P < 0.05) among treatments, as 45.62 2.89 nN m1 was found for chitosan and 31.15 0.148 nN m1 for chitosan + lauric acid. Addition of lauric acid decreased intermolecular interaction forces, leading to decreased surface tension.


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Chitosan is a polymer formed by beta (1–4) 2-amino2-deoxy-D-glucose monomer repetition, of polar nature. In adding lauric acid (nonpolar), weaker intermolecular interactions begin occurring, which results in reduced surface tension. There was no significant difference in apparent viscosity among the two coating formulas (P > 0.05), with an average of 0.175 0.005 Pa.s. Both solutions had a flow behaviour index (n) of 0.84, having thereby a non-Newtonian pseudoplastic behaviour as apparent viscosity decreases as applied strain rate increases (Schramm, 2006). Determination of vacuum application time

As per Table 1, there was significant variation in firmness of minimally processed pumpkins regarding different vacuum application times (P < 0.05), where 8 min presented least firmness. Regarding the thickness of chitosan edible coating applied to minimally processed pumpkins, there was significant variation (P < 0.05) among vacuum application times (Table 1). Vacuum application time of 2 min yielded coating thickness of 21,33 lm, thinner when compared to other treatments. Regarding water content, there was no significant variation (P > 0.05) between different vacuum application times, although all treatments lost humidity throughout shelf life, and this loss was greater on pumpkins treated with 2 min of vacuum application. The application lasting 4 min at 500 mmHg vacuum pressure was selected, because minimally processed pumpkins that underwent this period of vacuum application of chitosan coating presented no significant decrease in coating thickness, texture or water content. Chemical and physical characteristics Titratable acidity, potential of hydrogen (pH) and soluble solids content (SSC)

Vacuum impregnation method led, immediately after processing, to a decrease in pH (5.61 0.09) and a rise in acidity (0.165 0.04) of MMPs, when

compared to soaking technique (6.48 0.19 and 0.103 0.06, pH and acidity, respectively) (P < 0.05), likely due to a greater incorporation of coating solution (which is more acidic). According to Fito (1994), when vacuum is applied to a system, air from within the tissue flows to the exterior through pore spaces and such spaces are then filled with a greater amount of external solution (acid solution) due to the hydrodynamic mechanism. This does not occur in the soaking technique, once it does not cause air removal because of the absence of vacuum. Relating to coating composition and storing time, there was no significant difference (P > 0.05). It was found that neither methods nor different coatings altered soluble solids content, same for storing period (P > 0.05). It presented an average of 8.18% 0.07. Water content

The method of coating application did not influence water content in pumpkins (P > 0.05). However, pumpkins with an edible coating, either chitosan or chitosan + lauric acid, presented higher water content when compared to pumpkins with no coating (P < 0.05). Regarding storage period, all treatments exhibited water loss; nonetheless, samples with coating exhibited it to a smaller extent (Fig. 1). Thus, application of an edible coating of either chitosan or chitosan + lauric acid acted efficiently in preventing humidity loss. Although a few studies show that adding a lipid compound in coating formula enhances the barrier against water loss (Vargas et al., 2006), results of this study found that the addition of lauric acid was not enough to minimise the water vapour permeability of chitosan. No difference was found in water content (%) of minimally processed pumpkins treated with chitosan and chitosan + lauric acid (P > 0.05). Total carotenoids

Total carotenoids did not differ among the different coating application methods (P > 0.05), although it did differ among the different coatings applied (P < 0.05), with the control treatment having a lower

Table 1 Firmness, water content (%) of minimally processed pumpkins and chitosan coating thickness in different vacuum application times

Water content (%) Time (days) Time (min)

Firmness (N)

2 4 6 8

36.07 35.09 32.21 30.53

1.42a 0.45a 1.31a,b 1.67b

Thickness (lm) 21.33 24.17 24.25 25.41

0.47a 0.71b 0.44b 0.39b

0 86.25 85.85 85.54 86.42

16

0.62Aa 0.24Aa 0.56Aa 1.5Aa

79.32 81.63 81.34 82.60

0.53Ba 0.23Bb 0.74Bb 0.15Bb

Equal lowercase letters do not differ statistically at 5% significance by Tukey’s test.




average (0.678 0.06) than both the chitosan coating (0.760 0.06) and chitosan + lauric acid (0.750 0.05). Only those in the control group varied with storage time, indicating the efficiency of coating application. The complex structure of plant tissue protects carotenoids in natura; however, processing causes rupture of cellular structure, increasing surface area and exposing carotenoids to oxygen, light and oxidising enzymes, stimulating a series of degradation reactions (Rodriguez-Amaya, 2001). The application of edible coating creates a modified atmosphere with high CO2 contents and low O2 (Baldwin & Nisperos-Carriedo, 1995), decreasing oxygen availability and delaying carotenoid degradation by oxidation. Colour

Immediately after processing, pumpkins submitted to soaking method showed superior average numbers for coordinates L* (48.28 3.65 for soaking, 41.00 1.53 for VI), a* (28.65 2.10 for soaking, 25.60 1.75 for VI) and b* (68.40 2.04 for soaking, 61.88 3.34 for VI) when compared to pumpkins that underwent vacuum impregnation (P < 0.05). Moreover, after 16 days of storage, pumpkins presented significant difference for coordinates, where L showed increase over time (55.24 0.54 soaking, 48.92 0.73 VI), and the coordinates a* (22.26 0.37 soaking, 20.57 0.07 VI) and b* (47.87 1.07 soaking, 43.82 1.57 VI) presented decrease over storage period. The increase in luminosity over storage period is linked to whitening, which is a result of partial dehydration of surface cells due to processing. The decrease of coordinates a* and b* indicates a decrease in red (a*) and yellow (b*) colouring, frequent in minimally processed produce due to previously endured damage. This damage may lead to oxidation and subsequent

degradation of carotenoids, which grants pumpkins their red and yellow colouring (Alves et al., 2010). Furthermore, the phenomena which occur in VI (expansion and liberation of gas stuck in pores and incorporation of components after atmospheric pressure restoration) can result in higher reflectance of light beams, leading to a reduction of luminosity in the produce (Derossi et al., 2010). Puente et al. (2009) found that apple slices become darker after VI, corroborating that the process reduces fruit brightness due to the removal of oxygen from the pores, causing an increase in reflectance. DE measures the colour difference among control and processed groups. The greater the DE, the greater the total difference between processed produce when compared to the control group. According to Adekunt et al. (2010), differences in perceivable colour can be classified analytically as very distinct (DE >3), distinct (1.5 < DE