The effects of added sugars and alcohols on the induction of crystallization and the stability of the freeze-dried peki (Caryocar brasiliense Camb.) fruit pulps

LWT - Food Science and Technology 43 (2010) 934–941

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The effects of added sugars and alcohols on the induction of crystallization and the stability of the freeze-dried peki (Caryocar brasiliense Camb.) fruit pulps Cibele Cristina de Oliveira Alves, Jaime Vilela de Resende*, Moˆnica Elisabeth Torres Prado, Rafael Souza Ribeiro Cruvinel Department of Food Science, Federal University of Lavras, P.O. Box 3037, 37200-000 Lavras, Minas Gerais, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 July 2009 Received in revised form 26 January 2010 Accepted 29 January 2010

Peki (Caryocar brasiliense Camb.) is a Brazilian fruit with an extremely high b-carotene content, but the b-carotene is unstable under dry storage conditions. This work reports on the product development and stability of freeze-dried peki fruit pulp. Freeze-dried products were made by adding alcohols (ethanol and isopropyl alcohol; at concentrations of 0, 5 and 10 mL/100 mL of extract) and sugars (sucrose and fructose; at concentrations of 0, 5 and 10 g/100 mL of extract) to the peki fruit pulps followed by freezedrying. Scanning electron microscopy was used to analyze the microstructure of the freeze-dried products by visualizing the crystallized forms. The product hardness and total carotenoid content following the different treatments were measured using a texture analyzer and a spectrophotometer, respectively. The stability of these foods was evaluated by their water sorption during their storage in various relative humidity environments at 25  C. There were characteristic differences in their hygroscopic behaviors. The pretreatment with sucrose and ethanol improved the freeze-dried product and yielded a lower number of collapsed structures. Changes during the storage were observed. The pulp pretreated with sucrose was amorphous and metastable, but the drying process was accelerated by the presence of alcohol (mainly ethanol), which resulted in protected structures without any sign of collapse. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Freeze-drying Crystallization induction Carotenoids Water sorption Collapse

1. Introduction Peki (Caryocar brasiliense Camb.) is a typical cariocacea that can be found evenly distributed throughout the southeastern, central western and northeastern regions of Brazil, where it represents the main source of income for the population. Recently, the fruit has gained nutritional and economic importance, and its commerce has expanded beyond the Brazilian borders as it is now exported to other countries, including Australia. Peki fruit is consumed for its unique flavor and aroma as well as its high nutritional value. Peki has an extremely high b-carotene content (Ribeiro, 2000) that is not stable during storage. The importance of some carotenoids as precursors of vitamin A in human nutrition is well known, and b-carotene is the most powerful of these provitamins. This is significant because vitamin A deficiency is considered a nutritional problem of populations in the poorest regions of Brazil. Peki oil is another sub-product that is extracted from the fruit pulp. According to Vilas Boas (2004), peki pulp is composed of * Corresponding author. Tel.: þ55 35 3829 1659; fax: þ55 35 3829 1401. E-mail address: [email protected]fla.br (J.V. de Resende). 0023-6438/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.029

49.2% moisture, 20.5% lipids, 4.2% protein, 18.9% carbohydrate, 6.8% fibers, 9.4% minerals and 0.9–2.0% titratable acidity. With its widespread use, peki is an important fruit consumed by a large portion of the Brazilian population, and all parts of the peki fruit are used for various purposes. However, peki fruit harvests occur only during the months of November to February. This is a limiting factor in the commercialization of and access to the peki fruit. Peki dehydration would be an excellent means to preserve the fruit and secure its commerce in the periods between harvests. Stable, edible products from the peki pulp (internal mesocarp) would also allow for its use in varied market niches and increase the fruit’s aggregate value as a flavoring and an ingredient in beverages, ice cream, bread and confectioneries. Freeze-drying results in products with a higher flavor quality compared to those dried by conventional methods (Krokida & Philippopoulos, 2006; Marques, Prado, & Freire, 2009). However, the freeze-drying process causes certain changes that produce amorphous and hygroscopic structures. These structures are sensitive to changes in their physical, chemical and microbiological characteristics, which reduce the shelf life and stability of the product (Fabra, Talens, Moraga, & Martinez-Navarrete, 2009; Telis & Sobral, 2001).

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The physical state of amorphous materials may change from a solid glassy state to a liquid-like rubbery one when the glass transition temperature (Tg) is reached. Food powders containing amorphous sugars experience stickiness and caking when the powder is exposed to temperatures above the powder’s glass transition temperature (Tg), which is function of the moisture content or water activity of the powder. Foster, Bronlun, and Paterson (2006) have proposed and confirmed that the glass transition-related flow changes (sticking, crystallization and collapse) and the rate of change of the cohesiveness are directly dependent on the amount that the glass transition temperature (Tg) is exceeded (T  Tg). When the moisture evaporates during the freeze-drying process, the product becomes porous in nature, and the solid network should be able to hold this porous structure (Khalloufi et al., 2000). If the temperature of the dehydrating porous product is above the glass transition temperature (Tg), the viscosity of the solid material may not be enough to support the structure, and collapse or shrinkage occurs. This structural shrinkage leads to poor aroma retention, poor rehydration characteristics and an uneven dryness. Foods that have high sugar contents, such as fruit juices, will have lower collapse temperatures (Bhandari & Howes, 1999; Boonyai, Bhandari, & Howes, 2004; Foster et al., 2006; Franks, 1998; Krokida, Karathanos, & Maroulis, 1998; Roos & Karel, 1991; Venir, Munari, Tonizzo, & Maltini, 2007). The product properties may be substantially affected by the form of the solid produced, particularly when it is in the amorphous or crystalline form (Chiou, Langrish, & Braham, 2008). For sugars, the crystalline state is the most stable and generally has the lowest free energy. The crystallization process is dependent on the oversaturation of the system, which is dependent on the temperature, concentration and sugar solubility. In the case of the freeze-drying process, an alternative to induce the crystallization is the addition of organic solvents such as alcohols before the freezing process, which can reduce the sugar solubility and to promote its crystallization via the oversaturation of the system (Almeida & Cal-Vidal, 1997; Singh, Shah, Nielsen, & Chambers, 1991). Another alternative to aid the induction of crystallization is the addition of small sugar crystals that would act in the heterogeneous nucleation process. The addition of sugar during the production of freeze-dried fruit pulp also has the function of holding onto the flavor compounds that are otherwise lost during processing. Carotenoids prevent the formation of free radicals in the body and, thereby, prevent tumors and the development of cardiovascular diseases. However, carotenoids are extremely susceptible to oxidative reactions because they have a large number of unsaturated bonds. In fruits and vegetables found in nature, the cellular structure and protein complexes provide a certain degree of stability. During some stages of dehydration, the ultra-structure and complexes can be broken. In this process, carotenoids are exposed to adverse factors and are degraded. Thus, the use of dehydration techniques and composite additions, which guarantee the stability of the carotenoids, is extremely important to assure a quality product that will be accepted in the market. The objectives of the current study were: (i) to develop a food product from freeze-dried peki pulps (internal mesocarp); (ii) to analyze its crystalline microstructure using scanning electron microscopy; (iii) to characterize the hardness of the product using a texture analyzer with an axial compression test; (iv) to characterize the preservation of the carotenoid contents of the freezedried peki pulps made with the addition of sugars and alcohols using a spectrophotometer; and (v) to determine and compare the product water sorption behavior as a function of the storage time for the different formulations.

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2. Materials and methods 2.1. Preparation of the extracts and the pre-freezing treatments Peki fruits (C. brasiliense Camb.) were harvested in Sete Lagoas/ MG (Brazil). Fruits of uniform size, shape and ripeness were washed with water, sanitized with sodium hypochlorite (200 mL/L), packaged in opaque plastic bags and stored under refrigeration at 10  C and 90% relative humidity for 24 h. The external mesocarp was manually removed with a knife and discarded. The pulps were prepared by cutting the internal mesocarp with a knife and grinding for the extract preparation; the final pulp concentration was 30 g/100 mL of extract. Sugars (fructose P.A., 99.5% – Synth, Brazil or sucrose P.A., 99.8% – Isofar, Brazil) and/or alcohols (ethanol, 99.3% – Isofar, Brazil or isopropyl alcohol, 99.5% – F. Maia, Brazil) were added to the fruit pulp at concentrations of 5 or 10 mL/ 100 mL of extract, and distilled water was added to the solution to bring the final volume to 100 mL of extract. The extracts were homogenized, filtered, placed in glass plates and frozen with static air (deep freezer; REVCO, Asheville, USA) at 60  2  C. The frozen extracts were carried to the pilot freeze-drier (Liobras-L101, Sa˜o Carlos, Brazil), which was maintained at 40  C with a vacuum pressure of 0.0998 kPa (0.998 mbar), where they remained for an average time of 72 h. During the secondary stage of drying, the product reached a final temperature of approximately 35  C. The freeze-dried products were then broken, milled and homogenized using a Turratec grinder (Tecnal, model – TE-102, Brazil). To determine the particle size range, we used a test sieving procedure with a machine (Produtest, model T, Telastem, Sa˜o Paulo – Brazil) for 5 min with a 1.6 mm vibration amplitude, 3600 vibrations/min and 2.36 mm, 1.18 mm, 0.850 mm and 0.425 mm sieve sizes (Brazilian Association of Technical Normalization, sieve sizes: ABNT 8, 16, 20, 40). 2.2. Microstructural analyses The freeze-dried products were fixed with double-sided carbon tape onto an aluminum support (stubs) that was sputter-coated under vacuum with a thin film of metallic gold using a Bal-Tec model SCD 050 evaporator (Balzers, Liechtenstein). A Nano Technology Systems (Carl Zeiss, Oberkochen, Germany) model EvoÒ 40 VP scanning electron microscope was used with an accelerating voltage of 20 kV and a working distance of 9 mm to obtain the digital images using the Leo User Interface software at varying magnifications. The images were processed using Corel Draw 14 Photo paint Software. 2.3. The total carotenoid contents The freeze-dried peki pulps, which were obtained from the internal mesocarp, were analyzed for their total carotenoid contents. A weight portion (1–10 g) was used to measure the total carotenoids. The carotenoids were extracted with isopropyl alcohol and hexane. The total carotenoid content was determined by measuring the absorbance at 450 nm in a spectrophotometer (Varian, model Cary 50 Probe, Australia) using the extinction coefficient of 2500, according to the procedure of the Adolph Lutz Institute (1985) with modifications. The results were expressed as the ratio of the carotenoids (mg) to the freeze-dried product (g) (Lima et al., 2005; Rodriguez-Amaya, 2001). 2.4. The hardness of the freeze-dried peki pulps A Texture Analyzer (TA-XT2i, Stable Micro Systems, England) fitted with a flat-ended cylinder probe (0.6 cm diameter) was used

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to measure the hardness of the freeze-dried pulp. The hardness of the freeze-dried pulps was measured using an axial compression test that was applied to the samples in a cylindrical cup (2.0 cm diameter; 5.0 cm height) for 5 s with a test speed of 1.0 mm/s. The cylindrical cup was filled with the sample to keep the test volume constant. The measurements were carried out with five replicates and with samples whose water activities were those obtained immediately after the freeze-drying process.

2.5. The water sorption behavior analysis The appropriate environments for the determination of the water sorption behavior of the freeze-dried products were created in containers that were closed hermetically using saline solutions of varying saturation levels with the following water activities (Aw) at 25  C: LiCl, 0.11; CH3COOK, 0.22; MgCl2$6H2O, 0.32; Mg(NO3)2$6H2O, 0.53; NaCl, 0.75; KCl, 0.8; and BaCl2, 0.90. After freeze-drying, the samples (5.0 g) were weighed, distributed on glass plates (5.0 cm diameter, 1.5 cm height) as a single layer and carried to containers with saturated saline solutions. The data for the analysis of the water adsorption dynamics of the freeze-dried products were obtained at 25  C for each environment of controlled relative humidity. The sorption isotherms were built using the water activity measurements that were made using a dew point hygrometer (Aqualab, Decagon; model 3TE, Pullman, Washington, USA) with a time interval of 24 h on the first and second days. The measurements were made over the next days at a time interval of 48 h until equilibrium was established. The water sorption behaviors were plotted on a graph relating the product water activity as a function of the equilibrium relative humidities of the environments and the storage time.

2.6. The experimental delineation and statistical analysis The treatment effects on the total carotenoid contents and product hardness as a function of the sugar and alcohol concentrations were evaluated using the surface response methodology (Statistica software, version 7.0, StatsoftÒ). The non-linear regression was used to fit the curves and to evaluate the hygroscopic behavior of the freeze-dried pulps. The experimental setup with the central and axial points is presented in the Table 1. The following criteria were used to evaluate the experimental data: 1) the significance of the parameter estimates (probability level of 5%); 2) the determination coefficients (R2) between the observed responses and the estimated values for the fitted model.

Table 1 Experimental setup (combination of factors 1 and 2). Factor 1. Sugar

Level Sucrose

Fructose

2. Alcohol

Ethanol

Isopropyl alcohol

-

Concentration Concentration Concentration Concentration Concentration Concentration

(0 g/100 mL)) () (5 g/100 mL) (0) (10 g/100 mL) (þ) (0 g/100 mL) () (5 g/100 mL) (0) (10 g/100 mL) (þ)

-

Concentration Concentration Concentration Concentration Concentration Concentration

(0 mL/100 mL) () (5 mL/100 mL) (0) (10 mL/100 mL) (þ) (0 mL/100 mL) () (5 mL/100 mL) (0) (10 mL/100 mL) (þ)

3. Results and discussion 3.1. Scanning electron microscopy The analysis of the particle surfaces from the peki fruit pulps obtained by freeze-drying was carried out at a three-dimensional level through electronic microscopy and the electromicrographs are presented in Fig. 1. Fig. 1A refers to the systems without sugar but with added ethanol, and it demonstrates the amorphous structures, high bulk porosity and strong attraction and adherence of the smaller particles to the surface of the larger particles. One example of a system that involved the pretreatment with only sucrose is presented in Fig. 1B. It was verified that the particles were larger and strongly attracted to each other, and the typical structures that characterized an amorphous sugar state could also be observed. Fig. 1C shows an electromicrograph of the systems with both sucrose and ethanol. In these systems, the particles were uniform and did not strongly adhere to each other, verifying that the set contained scattered particles. Comparing the Fig. 1A–C, it is clear that the combination of sucrose and ethanol was effective at inducing the formation of crystalline structures characterized by a lower bulk porosity without a strong interaction among the particles. These features indicate that crystallization was induced during the freeze-drying process, and it was dependent upon the combination of ethanol and sucrose. The pretreatments that produced the structures characteristic of an amorphous sugar state also are show in the electromicrograph in Fig. 1D for samples that were pretreated with fructose and isopropyl alcohol. A larger agglomerate with strong interactions and inter-particle adherence was observed in these condition. The non-interacting particles formed during the drying process could reduce the stickiness phenomenon because crystalline sugar has a low water sorption potential. Some reports have indicated that adding some specific substances to a sugar solution before drying can result in some degree of crystallization. According to Hartel (1993), the addition of a second solvent in which sugar is not soluble and a temperature decrease favor oversaturation, and a solid phase forms starting from the original solution of the system. There are several published works on the induction of sugar crystallization, but most of them used organic solvents. Morr and Lim (1970) showed that the solubility of lactose decreases with the length of the alcohol chain. Ethanol and isopropyl alcohol are totally miscible in water because the strengths of their intermolecular interactions are similar to that of water, and, thus, they can compete with the sugar molecules for solvent interaction. 3.2. The hardness and particle size of the freeze-dried peki pulps The compressibility characteristics of the freeze-dried peki fruit pulps with the addition of sucrose and ethanol or fructose and isopropyl alcohol are depicted in Fig. 2. The height of the peak indicated the degree of hardness, and the width of the peak indicated the resistance of the samples during the compression test. Fig. 3 shows the response surface obtained using the quadratic model after the experimental hardness measurement of freezedried pulps pretreated with sucrose and alcohol. The hardness of the sucrose-pretreated freeze-dried pulps was not influenced by the nature or concentration of the tested alcohols, as the surfaces were located in nearly the same plane. The results for the fructose-pretreated samples as a function of the alcohol and sugar concentrations are presented in Fig. 4. Increases in the fructose concentration resulted in an increase of the hardness of the freeze-dried pulps. The effect was notable for the ethanol-treated samples.

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Fig. 1. Electromicrographs of the freeze-dried peki pulps following the various pretreatments: A) 0 g/100 mL sugar and 10 mL/100 mL ethanol; B) 5 g/100 mL sucrose; C) 10 g/ 100 mL sucrose and 5 mL/100 mL ethanol; and D) 10 g/100 mL fructose and 10 mL/100 mL isopropyl alcohol.

Collapse is generally observed in freeze-dried materials as a result of the plasticization of the concentrated solids; it is a consequence of viscous flow. Only a small amount of water is required to collapse such products. An immediate consequence of stickiness and/or collapse is that the particles become self-adhesive (cohesive) and slowly flow under gravity to form a sintered bed (Boonyai et al., 2004). Visibly, the freeze-dried peki pulps that were pretreated with fructose and ethanol (Fig. 4) or isopropyl alcohol (data not shown) were amorphous and had cohesive features. The pulps treated with sucrose plus ethanol were easier to handle, and this treatment did not induce cohesive features. The treatments with sucrose and

ethanol (Fig. 3) or with sucrose and isopropyl alcohol had lower values of hardness than those presented in Fig. 4. The higher values of hardness may be related to the microstructure characteristics of the peki freeze-dried pulps as illustrated in Fig. 1A, B and D, which show the characteristic amorphous particle surfaces and strong binding. A decreased cohesive force is related to the characteristics of the particles. The analysis of the particle size through the sieve showed that about 80 g per 100 g of the freeze-dried product had particle sizes lower than 1.20 mm for the treatments with ethanol and sucrose. For ethanol and fructose, this value was about 35 g per 100 g of freeze-dried product. According to Eduardo and Lannes (2007), samples with a higher percentage of small particles are more compactable (had a smaller compaction force). The samples

Fig. 2. Examples of the compression profiles of freeze-dried peki pulps characterized using a texture analyzer.

Fig. 3. Effect of ethanol and sucrose pretreatment on hardness of freeze-dried peki pulp (R2 ¼ 93.5%).

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Fig. 4. Effect of ethanol and fructose pretreatment on hardness of freeze-dried peki pulp (R2 ¼ 89.5%).

drying process, the sugar generates instability in the product and makes it more vulnerable to deteriorative reactions. This is because sugar interacts or competes chemically with other system components that damage the carotenoid extraction process and intervene with the spectrophotometer readings. The total carotenoids and vitamin A are lost during the dehydration process from foods that are rich in these composites. Although freeze-drying is a dehydration method that aims at preserving the functional and sensorial integrity of the food, the loss of carotenoids is a preoccupying factor in this process. Independent of the chosen processing method, the carotenoid degradation increases with the time, temperature, size and disintegration of the food particles. The stability widely varies with the processing and storage, and it is dependent upon the temperature, oxygen availability, light exposure, water activity, acidity, metal presence and the proper structure of the food. The degradation primarily occurs if there is no structural stability in the dried product. Reductions in the processing time, the temperature and the time between the cut, disintegration and processing improve the carotenoid retention significantly. Therefore, processing the foods at a low temperature and for a short period of time is a good alternative (Rodriguez-Amaya, 1993).

with smaller particles showed more homogenous particles with a better distribution and a low inter-particle interaction. The treatment with fructose and an alcohol resulted in greater values of cohesive force than the treatment with sucrose and an alcohol. 3.3. Analyses of the carotenoid contents Fig. 5 depicts the response surfaces fitted by a non-linear regression (quadratic) for the total carotenoid contents of the freeze-dried peki pulps pretreated with sucrose as a function of the ethanol (Fig. 5A) or isopropyl alcohol (Fig. 5B) concentrations after processing. Fig. 6 depicts the response surfaces for the total carotenoid contents of the freeze-dried peki pulps pretreated with fructose as a function of the ethanol (Fig. 6A) and isopropyl alcohol (Fig. 6B) concentrations after processing. For the systems using sucrose and ethanol (Fig. 5A), sucrose (P < 0.01), ethanol (P < 0.05) and their interactions (P < 0.05) significantly influenced the total carotenoid contents of the freezedried peki pulps. For the systems using sucrose and isopropyl alcohol (Fig. 5B), the factors with a significant influence were sucrose, isopropyl alcohol and their interactions (P < 0.01). In the systems consisting of fructose and ethanol or isopropyl alcohol (Fig. 6A and B), fructose and the interactions of fructose and the alcohol were significant (P < 0.01). The highest total carotenoid contents were found in the treatments with alcohol and no sugar. In these samples, the alcohol concentration influenced the carotenoid preservation. The results show that the total carotenoid contents varied proportionally with the concentration of the solvent used in the pretreatments. Considering the pretreatments as solvent extraction processes, the results demonstrated that the alcohol increased the effectiveness of the carotenoid extraction. Therefore, the higher alcohol concentrations had more interactions with the carotenoids. Moreover, the alcohol influenced the freeze-dried pulp stability, preventing losses occurring due to structural collapse. With very few exceptions, carotenoids are insoluble in water and soluble in organic solvents, such as acetone, alcohol, ethyl ether, chloroform and ethyl acetate (Rodriguez-Amaya, 2001). A different behavior is observed in the treatments with sugars (Figs. 5 and 6). Increasing the sugar concentrations in the freezedried peki pulps reduces the total carotenoid contents. Beyond contributing to the formation of amorphous structures during the

Fig. 5. Results of the total carotenoid contents (mg/g) of the freeze-dried peki pulps as a function of the sucrose concentration (g/100 mL) and the (A) ethanol concentration (mL/100 mL) (R2 ¼ 85.4%) or the (B) isopropyl alcohol concentration (mL/100 mL) (R2 ¼ 90.5%).

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content is low in the region with the low water activity. The dissolution of the sugar occurs at the high water activities when the crystalline sugar is converted into an amorphous sugar. The amount of adsorbed water increases greatly after this transition due to the increase in the number of adsorption sites upon breakage of the crystalline structure (Marques, Ferreira, & Freire, 2007). Systems consisting of ethanol and fructose (Fig. 7B), isopropyl alcohol and sucrose (Fig. 8A) and isopropyl alcohol and fructose (Fig. 8B) exposed to the equilibrium relative humidity lower than 40% had an initial fast increase in the solid water activity, followed by a reduction and leveling. For the other treatments, the initial reduction was followed by a new increase in the solid water activity; this occurred primarily in the systems with isopropyl alcohol (Fig. 8A and B).

Fig. 6. Results of the total carotenoid contents (mg/g) of the freeze-dried peki pulps as a function of the fructose concentration (g/100 mL) and the (A) ethanol concentration (mL/100 mL) (R2 ¼ 89.8%) or the (B) isopropyl alcohol concentration (mL/100 mL) (R2 ¼ 92.9%).

3.4. Water sorption of the peki freeze-dried pulps Changes in the water activities (Aw) of freeze-dried peki pulps as a function of the equilibrium relative humidity of the environments and the storage time are shown in Fig. 7 for the systems with ethanol and sugars (sucrose and fructose) and in Fig. 8 for the systems with isopropyl alcohol and sugars (sucrose and fructose). Fig. 7A shows that the addition of ethanol at concentrations of 5 mL/100 mL (Aw ¼ 0.11) or 10 mL/100 mL (Aw ¼ 0.06, data not shown) with sucrose yielded freeze-dried products with lower initial water activity values than ethanol at concentrations of 5 mL/ 100 mL (Aw ¼ 0.19, Fig. 7B) or 10 mL/100 mL (Aw ¼ 0.17, data not shown) with fructose. However, adverse effects were observed for the fructose treatments. The initial values of the water activity for the freeze-dried peki pulps treated with fructose and isopropyl alcohol presented the highest values (Aw ¼ 0.15, Fig. 8A and Aw ¼ 0.25, Fig. 8B). In the environments with a low relative humidity, the water activities of the systems quickly increased in the first days. At the low water activities, water can be adsorbed only to the surface hydroxyl groups of the crystalline sugar. Therefore, the moisture

Fig. 7. Water activities of freeze-dried peki pulps as a function of the equilibrium relative humidity of the environments and storage time for systems with ethanol: (A) 5 mL/100 mL ethanol and 10 g/100 mL sucrose; and (B) 5 mL/100 mL ethanol and 10 g/100 mL fructose.

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Fig. 8. Water activities of freeze-dried peki pulps as a function of the equilibrium relative humidity of the environments and storage time for systems with isopropyl alcohol: (A) 10 mL/100 mL isopropyl alcohol and 5 g/100 mL sucrose; and (B) 10 mL/ 100 mL isopropyl alcohol and 5 g/100 mL fructose.

The water activity increase followed by the reduction and leveling (Figs. 7B, 8A and B) was attributed to the transformation of the amorphous sugar in the freeze-dried pulps to the crystalline state. The great increase in the water activity in the first days of the equilibrium period was attributed to the water absorption between the randomly spaced molecules in the non-crystalline system, specifically the amorphous sugar. Part of this water content was then liberated after the formation of the crystalline sugar (Lai & Schmidt, 1990). For the systems with higher equilibrium relative humidity environments (ERH) and greater storage time, pretreatment with sucrose at concentrations of 5 g/100 mL with ethanol at concentrations of 5 mL/100 mL (Aw ¼ 0.78, ERH ¼ 0.9, Fig. 7A) or 10 mL/

100 mL (Aw ¼ 0.75, ERH ¼ 0.9, data not shown) resulted in lower water activities on the final day of storage. Freeze-dried pulps obtained from pretreatment with fructose at concentrations of 5 g/ 100 mL exhibited higher water activity values (Aw ¼ 0.87, ERH ¼ 0.9, Fig. 7B), especially when 10 mL/100 mL isopropyl alcohol was added (Aw ¼ 0.88, ERH ¼ 0.9, Fig. 8). The trends of the results in different equilibrium relative humidity levels indicate that systems consisting of sucrose and ethanol yield freeze-dried pulps that were more stable with lower water sorption during storage. The interactions between the molecules and the contact area justify the observed phenomena. The sucrose molecules can interact more than fructose, which has a shorter chain. Moreover, ethanol is an alcohol with a linear chemical structure while isopropyl alcohol has a ramified one. Therefore, interactions are easier between the molecules of sucrose and ethanol. Crystalline products have the disadvantage of a low porosity and are also harder to dissolve compared to amorphous ones, although crystalline products are more flowable than amorphous ones and thus are easier to handle (Chiou et al., 2008). The bulk porosity is greatly affected by the freeze-drying process (Tsami, Krokida, & Drouzas, 1999), and the observed differences in the moisture sorption capacity (Figs. 7 and 8) can be explained by the differences in the bulk porosity and pore size of the dried materials. When the treatments did not include sugars, the freeze-dried peki fruit pulp had an amorphous structure and was highly porous (Fig. 1A), absorbing more water than the pulps made with the sucrose and ethanol combination (Fig. 1C). Bensouissi, Roge, and Mathlouth (2009) noted that the nucleation of sugars depends on their solubility, which affects their viscosity and, consequently, their diffusivity in solution. Moreover, nucleation can be influenced by the stability of the bonds established with water. The dissociation of water and its removal from the vicinity of the crystal surface is a rate-limiting step in crystal growth and is responsible for the increase of the nucleation induction time. Proteins, sugars and other polyhydroxy compounds are often added during the freeze-drying process to serve as lyoprotectants. Generally, these protective agents must remain at least partially amorphous to adequately protect compounds during the freezing and drying. While these sugars and polyhydroxy compounds serve to protect the food, they tend to pose a problem for the freezedrying process because these amorphous systems have low collapse temperatures (Franks, 1998; Kasraian & DeLuca, 1995). The excipients used in the formulation typically have a low collapse temperature, which dictates the use of slow and conservative freeze-drying cycles. These slow and conservative cycles can be optimized by improving the rate of the mass transfer of water through the partially dried layer (Maia & Cal-Vidal, 1994). The mass transfer is usually expressed in the terms of resistance, with the dried product layer being responsible for almost 90% of the total resistance. 4. Conclusions The combination of sucrose and ethanol was effective at inducing the formation of crystalline structures with lower bulk porosity. The particles showed uniformity, but there was no strong adherence among the particles, verifying that the set of particles was scattered. In the treatments with fructose and isopropyl alcohol, amorphous sugars formed that were characterized by the formation of agglomerates. Higher values of hardness may be related to the microstructure characteristics of the peki freeze-dried pulps. Freeze-dried peki pulps pretreated with sucrose and alcohol (ethanol or isopropyl

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alcohol) had lower cohesive forces, which were related to the characteristics of the particles that were more homogenous, with a better distribution and low inter-particle interaction. Treatment with fructose and alcohol resulted in greater values of cohesive force than sucrose and alcohol. The physical stability of amorphous sucrose could be enhanced by additives that interfere with crystallization. The highest total carotenoid contents were found following pretreatment with alcohol and no sugar. In these treatments, the alcohol concentration influenced carotenoid preservation. Increasing the sugar concentrations in the freeze-dried peki pulps reduced the total carotenoid contents. The presence of alcohol during freeze-drying protected the primary structure and minimized changes in the shape of the product, with minimal shrinkage. In this case, we propose that the carotenoids became entrapped in the matrices that resulted from rapid dehydration. Freeze-dried pulps obtained from pretreatment with fructose at concentrations of 5 g/100 mL or 10 g/100 mL had higher water activity values, especially when 10 mL/100 mL isopropyl alcohol was added. The trends of the results from the different equilibrium relative humidity levels indicate that systems consisting of sucrose and ethanol yielded freeze-dried pulps that were more stable with lower water sorption during storage. The differences observed in the moisture sorption capacity can be explained by the differences in the bulk porosity and pore size of the dried materials. Freezedried peki fruit pulp that was pretreated with no sugar had amorphous structures and was highly porous; these absorbed more water than those pretreated with the sucrose and ethanol combination. Sucrose is amorphous and metastable, but the addition of alcohol (especially ethanol) can accelerate the drying process and result in the production of protected structures with no signs of collapse. Acknowledgements The authors wish to thank the Coordenaça˜o de Aperfeiçoamento de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) which financially supported this research and FAPEMIG for granting scholarships. References Adolph Lutz Institute. (1985). Normas analı´ticas do Instituto Adolfo Lutz – me´todos ´lise de alimentos. Sa˜o Paulo, p.v.1. quı´micos e fı´sicos para ana Almeida, L., & Cal-Vidal, J. (1997). Fruit sugar crystallization during freezing to reduce the hygroscopicity of freeze-dried products in powder forms. InICEF 7 – Proceeding of the 7th international congress on engineering and food, Vol. I (pp. 9–12). Bensouissi, A., Roge, B., & Mathlouth, M. (2009). Effect of conformation and water interactions of sucrose, maltitol, mannitol and xylitol on their metastable zone width and ease of nucleation. Food Chemistry. doi:10.1016/j.foodchem.2009.03.075. Bhandari, B. R., & Howes, T. (1999). Implication of glass transition for the drying and stability of dried foods. Journal of Food Engineering, 40, 71–79.

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