Influence of Collapsed Structure on β-carotene Stability in

Freeze-Dried Mangoes Nathdanai Harnkarnsujarit, Sanguansri Charoenrein

Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University,

Bangkok, Thailand (fagisscr@ku.ac.th)

ABSTRACT

β-carotene gives benefits to human health; however, it is unstable and its degradation is accelerated by

oxygen exposure. This study aimed to investigate the effects of collapsed and non-collapsed structures of

freeze-dried mangoes on the stability of β-carotene. Freeze-dried mango structures were manipulated using

three freeze-drying protocols. β-carotene content during storage was constantly monitored using high-

performance liquid chromatography. The β-carotene degradation followed first order kinetics. The results

showed that structural collapse decreased β-carotene degradation rate because the dense structures formed

prevented oxygen penetration through the solids. However, the occurrence of surface cracking together with

collapsed structure showed no protective effect on β-carotene. Such structure showed similar rate constant to

non-collapsed systems. The cracking on mango surface has a potential to allow high oxygen penetration

through the solids thus promoting β-carotene loss. These findings give benefit to the production of freeze-

dried fruits to increase storage stability of β-carotene.

Keywords: Structure; Collapse; Freeze drying; Mango; β-carotene

INTRODUCTION

Mango (Mangifera indica) is a prominent tropical fruit which is widely grown in Thailand. It contains high

amounts of β-carotene, a well-known carotenoid, which is responsible for the typical yellow color of the

mangoes. Moreover, β-carotene is very beneficial for human consumption as it is a provitamin A and

antioxidant. Among other carotenoids, β-carotene provides the highest vitamin A activity [1]. However, the

high degree of unsaturated hydrocarbons makes β-carotene sensitive to oxygen exposure and its degradation

mainly occurs via oxidation.

Freeze-drying produces high quality food of good appearance with well-retained flavor and nutritional

quality. A successful freeze-drying process retains the volume of the material which, in the dry state, is

usually highly porous, brittle and hygroscopic, but has excellent rehydration properties [2]. A typical freeze-

drying process consists of three stages: freezing, primary drying, and secondary drying. Freezing of food is

efficient to solidify most of the solvent which is typically water. Primary drying, or ice sublimation, begins

whenever the chamber pressure is reduced and the shelf temperature is raised as to supply the heat for ice

removal. Secondary drying is the stage where water is desorbed from the freeze concentrated matrix, usually

at both an elevated temperature and low pressure [3].

The porous structure formed during freeze-drying is of interest for the production of food powders, instant

foods, or additives because of the improved and quick rehydration properties of freeze-dried ingredients [4,

5]. However, the porous structure is not beneficial during long-term storage of freeze-dried products which

contain bioactive compounds. Such compounds are affected by deteriorative reactions depending on their

exposure to oxygen [5]. The oxidative stability of freeze-dried products is also reduced due to their porous

structure. Porosity increases the contact area and potential exposure to oxygen [6]. Petzold & Aguilera [7]

also stated that larger pores retained more occluded air in the matrix and the possibility of oxidation reactions

to occur in the final product was increased.

Krokida et al. [8] stated that the physical properties of freeze-dried materials depend upon the temperature

applied during freeze-drying. The aforementioned study further established the collapse of a plant material

after freeze-drying above its glass transition temperature (Tg). In the freeze-drying process, the removal of ice

by sublimation creates pores, the walls of which may collapse because either of the force of the surfaces or

gravity. The viscosity of the freeze-concentrated matrix, which is usually the major component of the pore

wall, prevents or retards collapse [9].

Collapse in freeze-dried matrices adversely affects properties of the freeze-dried materials. However, some

recent studies showed that collapse can reduce the rate of some chemical reactions, such as glucose loss via

Maillard browning [10], browning in lactose and lactose-protein matrices [11], lipid oxidation [12-15], and

slower release of some entrapped volatile compounds [16]. Acevedo et al. [17] observed a lower β-carotene

degradation in collapsed than in non-collapsed freeze-dried polymeric matrices. However, the effect of

collapse was studied at various storage conditions and there were no studies related to the pre-collapsed

structure prior to storage and β-carotene stability during storage in either model or real food systems. In this

study, the effect of pre-collapsed structure of freeze-dried mangoes prior to storage on β-carotene stability

was studied. Only a limited number of studies relating the degradation of bioactive compounds and food

structure including collapse are available. The objective of this study was to investigate the effects of freeze-

dried food structures, i.e.. collapsed and non-collapsed, on β-carotene degradation, in freeze-dried mangoes.

MATERIALS & METHODS

Raw material and chemicals

Ripe mangoes (cultivar ‘Nam Dok Mai’) were purchased from Chantaburi, Thailand. They were selected for

their homogeneity in size, weight, peel color, and floatation in a 4-5% NaCl solution. The total soluble solid

content of mangoes was determined to be in the range of 16–20 ۫ Brix.

Chemicals used for β-carotene determination, i.e., hexane, methanol, and acetonitrile (RCI Labscan,

Bangkok, Thailand), were of high performance liquid chromatography (HPLC) grade. Triethylamine and the

external standard, all-trans- β -carotene (β-carotene type II synthetic ≥ 95% crystalline; C40H56, HPLC) were

purchased from Sigma-Aldrich (St. Louise, U.S.A.).

Preparation of freeze-dried mango structure

Mangoes were washed, peeled, and cut into 1-cm cubes and freeze-dried (Freeze dryer Dura-Top/ Dura Stop

MP, Dura Dry MP FTS Systems™, Stone Ridge, NY, USA) with the application of the 3 freeze-drying

protocols, i.e., various freezing methods and freeze-drying temperatures (primary drying). The protocol 1 and

2 consisted of freezing at a shelf temperature of -35 °C, whereas mango cubes in protocol 3 were frozen by

immersion in liquid nitrogen prior to transfer to the freeze-dryer at a shelf temperature of -35 °C. Frozen

cubes of mangoes were kept at -35 °C for 120 min for ice formation. Then the mango samples were freeze-

dried using shelf temperatures of -40 °C (protocol 1 and 3) or -15 °C (protocol 2) (primary drying), with the

final product temperature (secondary drying temperature) set as 30 °C for all protocols. All freeze-dried

samples were stored in evacuated desiccators over silica gel to reduce water uptake for 3 days.

Characterization of freeze-dried mango structures

The external structures of the freeze-dried mangoes were determined with a Spot camera (Leica S8APO,

Leica Microsystems®, Switzerland), which was connected to a stereo light microscope (Leica CLS 150 XE

Leica Microsystems®, Switzerland) at a low magnification (10×).

The internal structures of freeze-dried mangoes were characterized with the application of a scanning

electron microscope (SEM) (JEOL JSM-6480LV, Tokyo, Japan). Cross-sectioned samples were affixed on

silver-painted sample holders and covered with a fine layer of gold in a sputter coater (PSI sputter coater, PA,

USA) under vacuum. The coated samples were photographed with a SEM at 5 kV.

Thermal transitions of mangoes

Thermal transitions of mangoes were determined with a differential scanning calorimeter (DSC). Mango

juice was extracted and determined for thermal transitions as described by Lowithun and Charoenrein [18].

Mango pulp (500 g) was blended and centrifuged at 4,500g for the duration of 15 min. The supernatant was

taken as mango juice. Sample juice (10-13 mg) was transferred into aluminium DSC pans and hermetically

sealed. Duplicate samples were scanned without annealing to locate apparent Tg and onset of ice melting

(Tm׳) by DSC (Perkin Elmer Pyris 1 DSC). Samples were cooled from 25 to -60 °C, held for 15 min, and

subsequently heated to 25 °C at 5 °C/min. The Tg and Tm׳ were measured after isothermal annealing at Tm׳-

1°C obtained from the first scan. Triplicate samples were scanned from 25 to -60 °C, held for 15 min,

subsequently heated to Tm1-׳ at 5 °C/min and held for 30 min. After that, samples were cooled to -60 °C,

held for 15 min and heated to 25 °C at 5 °C/min. Tg׳ values were taken from the midpoint of the Cp change

over the glass transition and Tm׳ from the onset temperature of the ice melting endotherm.

Stability of β-carotene in freeze-dried mangoes

Freeze-dried mangoes were stored at 11.3% and 22.5% relative vapor pressure (RVP) in desiccators over

saturated solutions of LiCl or CH3COOK at room temperature (25 ± 2 °C). Samples were powdered in an

aluminum foil pouch to prevent uptake of water. Duplicates samples were prepared from freeze-dried

materials to determine β-carotene contents with a HPLC during storage. β-carotene was extracted and

measured with a method adapted from Ferruzzi et al. [19]. Freeze-dried mango powder (1 g) was extracted

with 10 mL of methanol and hexane (1:1) with a homogenizer (IKA T10 basic, Ultra Turrax, Germany) for 2

min. The suspensions were centrifuged (Sorvall RC 5C Plus Superspeed Centrifuge, MN, U.S.A.) at 4,500 g

at 4°C for 10 min. The supernatant (hexane layer) was collected and the precipitant was re-extracted. The

collected hexane layer was dried by purging with N2 gas. Dried residues were diluted with 2 mL of hexane

prior to preparation into an HPLC amber vial to assess the β-carotene content. Each sample (20 µL) was

analysed in an HPLC column C18 (XTerra® RP18, 5µm, 3.9 x 150 mm) (Waters, Milford, MA, USA).

Methanol:acetonitrile (9:1) with 0.1% trimethylamine was used as a mobile phase at a flow rate of 1.5

mL/min. The β-carotene was analyzed using a UV diode-array detector (Model 600, Waters, Milford, MA,

USA) at a wavelength of 450 nm.

RESULTS & DISCUSSION

3.1 Thermal transition of mangoes

Mango juice samples showed two endothermic transitions prior to ice melting peak during the DSC scan

which were typical of sugar-containing materials [20]. The two transitions were interpreted in different ways;

however, the lower transition (T′g) was the glass transition of the freeze-concentrated solids phase; T′g was

obtained after annealing to obtain maximum freeze-concentration of solids. The second, higher temperature

transition (T׳m), showed the onset of ice melting.

Mangoes contain high amounts of small molecular weight sugars that are sucrose, glucose, and fructose

which gave the low T׳g of - 53.3 °C. The T׳m was at -38.0 °C. These thermal transitions, particularly the Tm’

controls ice melting, directly affect the freeze-dried product structure.

3.2 Influence of freeze-drying protocols on the freeze-dried mango structure

Figure 1 shows the external structures of freeze-dried mangoes. The collapsed structure was observed in

freeze-dried mangoes when frozen at -35 °C and freeze-dried at the shelf temperature of -15 °C (protocol 2)

and when frozen with liquid nitrogen and freeze-dried using the shelf temperature of -40 °C (protocol 3);

freezing at -35 °C followed by freeze-drying at the shelf temperature of -40 °C (protocol 1) gave non-

collapsed structure.

Figure 1. Stereomicrographs of freeze-dried mango surface using freeze-drying protocol 1: freezing at -35 °C and shelf

temperature of -40 °C (A), protocol 2: freezing at -35 °C and shelf temperature of -15 °C (B) and protocol 3: freezing by

immersion in liquid nitrogen and shelf temperature of -40 °C (C).

Mangoes freeze-dried by the application of protocol 1 underwent the freezing stages at temperatures higher

than T׳g (-53.3 °C) but drying at a temperature lower than T׳m (-38.0 °C). This observation was coincident

with the non-collapsed structures of freeze-dried mangoes (shelf temperature -40°C). The loss of product

structure or collapse during freeze drying typically occurs when the temperature of the subliming interface is

maintained above the “collapse temperature” [21] which is related to the T׳m. Sacha & Nail [22], with the aid

of a freeze-dry microscope, observed that the onset of structural collapse in frozen sugar solutions was

associated with the higher transition temperature or T m׳ . The result shows that the sample temperature

exceeded the T׳g; however, it was below that of T m' , thus no structural collapse occurred. Moreover, the

strength of mango cell walls and polymers also supported the physical structure [23]. The external structures

of the samples are shown in Figure 1A. The application of freeze-drying protocol 1 was proven successful for

the retention of the tissue structures of the mango samples.

The freeze-drying protocol 2 resulted in collapsed structures (Figure 1B), which was due to the melting of the

ice during freeze-drying. The ice formed in the samples during the freezing stage at -35 °C. Freeze-drying

was controlled by the shelf temperature of -15 °C, which was above the T m' . The ice could consequently

melt, and the samples become dried by evaporation from a partially freeze-concentrated state rather than by

(B)

(C) (A)

sublimation which could result in the loss of structure [21]. In addition, food matrices are plasticized by

unfrozen water, thus the viscosity of the freeze concentrated matrix was decreased. This was responsible for

the acceleration of the collapse during freeze-drying [9]. In addition, the result showed that the duration of

the drying stage of the freeze-drying protocol 2 exceeded that of protocol 1.

Mangoes frozen by liquid nitrogen prior to freeze drying at the shelf temperature of -40 °C (protocol 3)

resulted in collapsed structures, although the freeze-drying temperature of protocol 1 and 3 were identical (-

40 °C). This was an evident indication of the negative effects of liquid nitrogen freezing on the structural

consistency of mangoes. The result showed that after freezing in liquid nitrogen, the sample temperature

increased during the freezing stage in the freeze dryer to exceed T g' . Freezing in liquid nitrogen produced

small ice crystals and fine porosity after initial stages of the freeze-drying. This presumably increased

resistance for vapor removal and a higher sublimation temperature at the sublimation interface. In addition,

the liquid nitrogen freezing resulted in formation of a crust at the mango surface, which further prevented the

mass transfer during sublimation. Furthermore, the cracking on surface was observed in samples which were

frozen in liquid nitrogen (Figure 1C). When the internal portion of the water underwent phase transition, it

caused an internal stress as a result of expansion to the surface and once the resistance of the mango was

overcome, cracking of the mango surface occurred [24]. Chassagne-Berces et al. [25] also observed the

cracking of apple tissue frozen by immersion in liquid nitrogen. This indicated that a too fast freezing rate

provoked breakage of food surfaces.

Figure 2 shows the SEM micrographs of the internal structure of the freeze-dried mangoes. The protocol 1

rendered non-collapsed samples with porous internal structures as shown in Figure 2A. The collapse structure

caused by the application of protocols 2 and 3 revealed large gaps and dense dry layer structures because the

sublimation could not be achieved; the internal water vapor pressure caused melting and water was instead

removed by evaporation. This unfrozen water plasticized the dry layer formed at the beginning of drying and

the internal pressure contributed to formation of a large gap inside the structure (Figure 2B).

Figure 2. SEM images of cross-sectioned freeze-dried mangoes using freeze-drying protocol 1: freezing at -35 °C and

shelf temperature of -40 °C (A), protocol 2: freezing at -35 °C and shelf temperature of -15 °C (B) and protocol 3:

freezing by immersion in liquid nitrogen and shelf temperature of -40 °C (C).

3.3 Stability of β-carotene in freeze-dried mangoes

11% RVP

-1

-0.8

-0.6

-0.4

-0.2

0

0 2 4 6

Time (weeks)

Deg

rad

ati

on

ra

te (

ln[C

/Co

])

22%RVP

-1

-0.8

-0.6

-0.4

-0.2

0

0 2 4 6

Time (weeks)

Deg

rad

ati

on

ra

te (

ln[C

/Co

])

Figure 3. Degradation of β-carotene in freeze-dried mangoes as a result of different freeze-drying protocols during

storage at 11% (A) and 22% (B) RVP. ♦ protocol 1: freezing at -35 °C and shelf temperature of -40 °C, ▲ protocol 2:

freezing at -35 °C and shelf temperature of -15 °C and ■ protocol 3: freezing by immersion in liquid nitrogen and shelf

temperature of -40 °C.

Freeze-dried mangoes which were exposed to the three diverse freeze-drying protocols were kept at 11% and

22% RVP to determine β-carotene stability over time (Figure 3). Generally, storage at high RVP led to

(C) (B) (A)

(A) (B)

various changes in dried foods such as structural collapse and sugar crystallization. To exclude these effects,

inclusive structural transformations, the storage RVP was selected in accordance to the previous work which

showed that freeze-dried mangoes stored at 25 °C at 11% RVP were in the glassy state, whereas they became

rubbery at 22% RVP. However, no structural change including collapse was observed, thus both RVP were

selected for monitoring the stability of β-carotene in freeze-dried mangoes during storage.

β-carotene is naturally unstable in the presence of oxygen and light. After six weeks of storage, β-carotene in

samples freeze-dried by the application of protocols 1, 2, and 3 decreased approximately 57%, 43%, and 59

% respectively. The amount of the degradation at both RVP was similar. β-carotene degradation followed

first order kinetics which was in agreement with previous studies in both food and model systems, such as

carrots [26], and a trehalose matrix [13,27].

Table 1 Rate constant of β-carotene degradation in freeze-dried mangoes

Rate constant Freeze-drying

protocols 11% RVP 22% RVP

1 0.1473± 0.0076 a 0.1393± 0.0137 a

2 0.0907± 0.0014 b 0.0833± 0.0071 b

3 0.1418± 0.0189 a 0.1517± 0.0072 a

a,b Different letters in the same column show significant differences (p≤0.05)

Rate constants of β-carotene degradation at both storage RVP are shown in Table 1. Increased rate constants

indicated higher degradation rates. The results showed that the freeze-drying protocol 1 and 3 rendered

similar degradation rates. In contrast, protocol 2 decreased β-carotene degradation rate significantly (P<0.05),

which attributed to the collapsed structure of the freeze-dried mangoes. Structural collapse gave a dense

internal matrix (Figure 2B) which has been found to increase the stability of β-carotene and other carotenoids

in model systems. Prado et al. [13] observed that storage RVP at which structural collapse was found

increased the stability of encapsulated β-carotene in freeze-dried polyvinylpyrrolidone. Selim et al. [14] also

suggested that matrix collapse could decrease the degradation of saffron carotenoids. Similarly, Serris &

Biliaderis [15] observed the lower degradation kinetics of water-soluble beetroot pigments in a collapsed

matrix. Collapse of freeze-dried materials is associated with the loss of micropores and cavities through

which oxygen can diffuse, so collapse of freeze-dried materials builds a barrier against oxygen penetration

[14]. β-carotene is naturally more stable in the absence of oxygen. Contrarily, the higher rate constant in

samples freeze-dried with protocol 1 is explained by the high porosity (Figure 2A) which allowed a higher

rate of oxygen diffusion [13]. Surprisingly, structural collapse induced by liquid nitrogen renders no

protection form the degenerative effects on β-carotene. It is hypothesized that the cracking of sample surface

can have a potential to increase the oxygen exposure through food matrix, which results in the rapid β-

carotene degradation.

CONCLUSION

Mangoes freeze-dried at temperatures above T׳m had collapsed structures. Moreover, freezing by means of

liquid nitrogen not only resulted in collapsed structures, but also the cracking on surface of the dried

products. The results showed hat collapse reduced β-carotene degradation in freeze-dried mangoes because

the loss of matrix micropores and dense structure potentially prevented oxygen diffusion. The presence of

cracking concurrent with collapse did not help to decrease the degradation of β-carotene. The collapse

structures of freeze-dried materials give benefits on decreasing the degradation of oxygen sensitive bioactive

compounds which subsequently increase the health benefits to consumers.

ACKNOWLEDGEMENT

This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program

(Grant No. PHD/0224/2549) and Kasetsart University Research and Development Institute (KURDI). The

authors thank professor Yrjö H. Roos for the helpful suggestions and critical reviewing of this manuscript.

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