Abstract
Changes in quality of low-moisture conditioned pine nuts (Pinus gerardiana) under near freezing temperature storage were investigated. Nuts were adjusted to different moisture content (17.3% ± 0.42, 15.1% ± 0.26, and 13.3% ± 0.24) with 35°C, 20%RH, sealed in air-tight jars and stored at −3 and −1°C for 12 months. In general, free fatty acid content and peroxide value of the nuts increased, while sensory quality decreased during the storage. Low-moisture nuts had lower respiration, free fatty acid content, and peroxide value. Nuts stored at −3°C had less visible mold infection and browning percentage than at −1°C. Low-moisture treatment exerted a slight negative effect on texture, but had significant preservative effects on color, odor, and taste during storage. Overall, integration of low-moisture conditioning and near freezing temperature storage can be a promising non-chemical way for maintaining the postharvest quality and extending shelf-life of pine nuts.
Se investigaron los cambios en la calidad de piñones en condiciones de baja humedad (Pinus gerardiana), almacenados a temperaturas cercanas al punto de congelamiento. Éstos fueron tratados con un contenido de humedad diferente (17,3% ± 0,42, 15,1% ± 0,26 y 13,3% ± 0,24), con 35° C, 20% HR, sellados en frascos a prueba de aire y almacenados a −3 y −1° C durante 12 meses. En general, se constató que, durante el almacenamiento, aumentó el contenido de su ácido graso libre así como su valor de peróxido, disminuyendo su calidad sensorial. Los piñones almacenados con más bajo nivel de humedad presentaron niveles de respiración, ácido graso libre y valor de peróxido más reducidos, en tanto que aquellos que se almacenaron a −3° C mostraron niveles de infección de moho visible y porcentajes de oscurecimiento menores que los almacenados a −1° C. El tratamiento para bajar la humedad tuvo un efecto negativo menor en la textura y surtió efectos conservantes significativos en el color, el olor y el sabor durante el almacenamiento. En general, la integración del tratamiento de baja humedad con el almacenamiento a temperaturas cercanas al punto de congelamiento puede ser un método no químico prometedor para preservar la calidad post-cosecha y extender el periodo de conservación de piñones.
Introduction
Chilgoza pine nut (Pinus gerardiana W. ex D.) is traditionally consumed in various parts of the world as an important ingredient in desserts, sauces (e.g. in pesto), or salads. Though mostly distributed in the natural range of Afghanistan, Pakistan, India, and China, it is an important international trade commodity worldwide (Destaillats, Cruz-Hernandez, Giuffrida, & Dionisiet, 2010). Chilgoza pine nut is rich in unsaturated fatty acids (especially polyunsaturated fatty acids), which offers health benefits especially in relation to blood serum lipid profile (notably the decrease in undesirable low-density cholesterols, very low density lipoproteins and low density lipoproteins (Destaillats, Cruz-Hernandez, Giuffrida, & Dionisi, Citation2010; Venkatachalam & Sathe, Citation2006). As pine nut contains high amount of fats and unsaturated fatty acids it is prone to hydrolytic and oxidative rancidity which lead to losses of pine nut in storage (Cavalett, Delacruz, Ross, & Yamamoto, Citation1966; Kaijser, Dutta, & Savage, Citation2000). Deterioration of pine nut quality in storage is caused by metabolism of nuts and microorganisms, which depends on storage conditions: temperature, moisture content, and gas composition.
Much research has been carried out on the relationship between storage conditions and shelf-life of cereals and nuts products, and low-temperature storage and air-tight storage are regarded as effective systems (Maskan & Karatas, 1999; Martin et al., Citation2001; Rehman, Citation2006). The respiration of nuts and growth of moulds are inhibited by reduced O2 and increased carbon dioxide (CO2) concentrations within the storage atmosphere, a condition induced by the respiration of nuts and moulds. We noted that maize could be preserved by air-tight storage without refrigeration in the rural areas of developing countries while low-temperature storage of near freezing point was possible to store grains for long term without significant deterioration in quality (Fukai, Matsuzawa, & Ishitani, Citation2003; Quezada et al., Citation2006). Meanwhile, low-moisture storage as a postharvest treatment has proven beneficial to control respiration rate (Dillahunty, Siebenmorgen, Buescher, Smith, & Mauromoustakos, Citation2000), inhibit microbial growth (Genkawaa, Uchino, Inoue, Tanaka, & Hamanaka, Citation2008), and retard oxidative rancidity (Rajarammana, Jayas, & White, Citation2010).
However, to our knowledge, no information is available on the storage effects on pine nut of low-moisture conditioning and near freezing temperature storage. The objective of this study was to examine the effects of these treatments on quality parameters on Chilgoza pine nut, including moisture content, relative humidity, respiration rate, free fatty acid content, peroxide value, free amino acids content, incidence of visible mold, browning rate and sensory quality. With a view to better understanding how these treatments help to keep fresh, retard oxidation and extend shelf-life.
Materials and methods
Materials and designs of experiment
Chilgoza pine nuts (Pinus gerardiana W. ex D.) were used for the experiments. The nut samples were received with an average moisture content of about 17.3% ± 0.42 (wet basis) initially. After removing impurities and broken kernels, the original nuts were stored at −1°C overnight.
Since many researchers have reported on the superiority of low-moisture content of cereals and oils products in storage (Al-yahya, Citation2001; Genkawaa et al., Citation2008; Moreno, Rivera, & Badillo, Citation1998), hence, a moisture range of 13–17% was selected for this study. The following day, pine nuts were conditioned in a hot air oven at 35°C and 20% relative humidity for 3 and 7 h to bring the moisture contents to 15.1% ± 0.26 and 13.3% ± 0.24, respectively. The original nuts with 17.3% ± 0.42 moisture content served as control. Pine nuts of the same moisture treatment were placed in 5 L volume air-tight plastic jars with screw caps, about 2.5 kg each.
A differential scanning calorimeter (DSC 204 F1 Phoenix, Netzsch, Germany) was used for analysis of the ice point of pine nuts. The DSC instrument was calibrated with pure indium standard before analysis. Aliquots (5 mg) of each sample were hermetically sealed into aluminum pans and placed into the DSC. An empty sealed pan served as a reference and nitrogen was used as carrier gas. Liquid nitrogen was used for sample cooling before the runs. Each pan and the reference were cooled from 20°C to −50°C at 5°C /min, held for 5 min and then heated to 10°C with the same rate and held for 5 min. The ice point of pine nuts was determined to be about −4°C. In the food industry, pine nuts were commonly stored at 0 ± 1°C for several months, so we explored −3 and −1°C as storage temperatures with the reasoning that these temperatures above the ice point could thus avoid the damage caused due to freezing. The jars were placed at −3 and −1°C for 12 months. There were three replicates for each treatment. Physiological and quality parameters were determined after 0, 2, 4, 6, 8, 10, and 12 months.
Nuts sampling
At each of the specified sampling day (0, 2, 4, 6, 8, 10, and 12 months), triplicate nut samples were randomly chosen from each treatment and control group. Kernels were removed from shells and cut into small pieces using a sharp knife, then mixed well and stored in air-tight plastic container −20°C for subsequent analysis of various parameters except otherwise specified. All analyses were conducted in triplicate.
Determination of moisture content and relative humidity
Moisture content of the nuts was measured by AOAC standard method (AOAC, Citation1995). Samples (3 g) were dried to the constant weight at 105°C. Moisture content was expressed on a wet basis. The relative humidity of the head space air in the jars was measured by using a smart data recorder and a humidity sensor (RC-TH501B, Htctech Co. Ltd., Hangzhou, China).
Respiration rate measurement
Respiration rate was measured with method described by Vlamis and Davis (Citation1943), with some modifications. Unshelled pine nuts (500 g) were placed in an airtight desiccator at a given storage temperature for 10 h, with a culture dish at the bottom of the desiccator containing 10 mL 0.4% sodium hydroxide. The CO2 was absorbed by sodium hydroxide. The residue sodium hydroxide was determined by titration with 0.2% oxalic acid with Phenolphthalein as a pH indicator.
Determination of peroxide value and free fatty acid content
Pine nut oil was extracted from 5 g samples by a Soxhlet extraction apparatus using ether as a solvent for 10 h (AOAC, Citation1995). The solvent was removed with a rotary evaporator and the residue was used to determine the peroxide value. The peroxide value was determined according to the method described by Bora and Rocha (Citation2004). Weighed residue was transferred into 250 mL glass-stoppered Erlenmeyer flask. CH3COOH‒CHCl3 (30 mL) was added followed by 1 mL saturated potassium iodide solution; the samples were shaken for 0.5 min and placed in the dark for 3 min. To this mixture was added 1 mL 1% starch solution and 80 mL H2O. The reaction mixture was finally slowly titrated with 0.01 mol/L Na2S2O3 until blue color just disappeared. The blank was determined using the same system but with pure water instead of the extracted oil. Free fatty acids were determined by titration method of AOAC (Citation1995) as percent oleic acid.
Free amino acids content assay
Free amino acids content was determined according to Sun, Lin, Weng, and Chen, (Citation2006), on the basis of ninhydrin colorimetric method. Pine nuts (1 g) were blended and homogenized with 5 mL of 10% acetic acid. The homogenized sample was brought to a volume of 100 mL with ammonia-free distilled water. After centrifuging, 1 mL of the supernatant was transferred into a 20 mL test tube with stopper followed by 1 mL ammonia-free distilled water, 3 mL ninhydrin solution, and 0.1 mL of 1 g/L ascorbic acid. The mixture was kept in a boiling water bath for 15 min before 15 mL of 60% alcohol was added. Finally, the tubes were placed in an ice bath and the absorbance (570 nm) of the reaction mixture was measured with a UV-Vis spectrophotometer (TU-1810, Beijing PG instrument Co. Ltd., Beijing, China).
The percentage of visible mold and browning nuts measurement
Thirty grams of pine nuts were accurately weighed and the visible molded kernels were separated, weighed, shelled, and recorded in percent (Raei, Mortazavi, & Pourazarang, Citation2010); kernels with brown color or visible oil leakage were included in browning count. The percentage of visible mold and browning nuts was determined in three replicates.
Sensory evaluation
The sensory attributes that characterize pine nuts quality were determined. These attributes were color, odor, texture, and taste (Mexis & Kontominas, Citation2009). Samples were evaluated by a sensory panel of eight trained panelists. They were nonsmokers, without food allergies, sensitive in smell, with consumption experience in dried nut products. Pine nut kernels were placed in odorless, white plastic plates coded with random numbers, and evaluations were performed within 2 h in order to avoid quality changes. The panelists were instructed to consume the whole sample and rinse their mouths with distilled water in between sample evaluation. Scoring was carried out using a 10-point scale (8∼10 = very good, 6∼8 = good, 4∼6 = acceptable, <4 = bad) for evaluation of odor, texture, and taste, and for color descriptor, anchored with white, white-yellow, yellow-white, and yellow-brown (8∼10 = white, 6∼8 = white-yellow, 4∼6 = yellow-white and <4 = yellow-brown). A score of 4 was taken as the lower limit of acceptability for color, odor, texture, and taste.
Statistical analysis
Values were expressed as means ± standard deviations. Data were analyzed using SAS (Statistical Analysis System) software. Analysis of variance (ANOVA) and Duncan's multiple range tests were used to compare significance of the difference between the samples.
Results and discussion
Moisture content and relative humidity
The changes in moisture content of pine nuts during storage are shown in . There was no significant change (p > 0.05) in moisture content of each treatment throughout the storage period. These results were similar with the findings of Rehman, Habib, and Zafar (Citation2002) who had already found no change in moisture content during storage of maize grains under low temperature and a gradual decline in moisture content stored at higher temperature. The changes in relative humidity in headspace gas of the storage jars during storage at −3 and −1°C are shown in . Therelative humidity differed from jar to jar depending on the moisture content of the nut samples, and was maintained virtually stable during storage.
Figure 1. Changes in relative humidity of storage space and respiratory rate of pine nuts during storage at −3 and −1°C: ▪, 17.3% moisture content m.c.; ⧫, 15.1% m.c.; ▴, 13.3% m.c. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means
Figura 1. Cambios en la humedad relativa del espacio de almacenamiento y de la tasa de respiración de piñones, almacenados a −3 y −1° C: ▪, 17.3% contenido de humedad c.h.; ⧫, 15.1% c.h.; ▴, 13.3% c.h. Cada punto de datos es la media de tres muestras replicadas. Las barras verticales representan los errores estándar de las medias.
Table 1. Moisture content (%) of pine nuts during 12 months storage.
Tabla 1. Contenido de humedad (%) de piñones almacenados durante 12 meses.
Respiratory rate
Respiratory rate is an important physiological parameter during storage of pine nuts. Control of respiration is benefit to prolong the storage time of nuts. By measuring the rate of CO2 accumulation in the free air space, the changes in respiratory rate of pine nuts during storage are shown in . In initial storage period, respiratory rate in treated pine nuts declined rapidly as storage progressed. From the start of month 5, respiratory rate of nuts remained relatively constant for subsequent 4 months, and slightly increased from month 9 to the end of storage. It was observed that during the storage period, the respiratory rate of nuts with low-moisture content was always lower than that of high-moisture content nuts, while under the same moisture content, nuts stored at −3°C had lower respiration as compared to nuts at −1°C. These results were consistent with those reported previously by Dillahunty et al. (Citation2000) and Nakakita and Ikenaga (Citation1997), who showed respiratory rate of rice reduced as the temperature decreased and increased as the moisture content increased. Thus, there was an interaction between moisture content and temperature on respiratory rate, the response of respiration to temperature was dependent on moisture content. Also, from the present study, low-moisture content nuts (13.3% ± 0.24) at −3°C showed a significantly lower level (p < 0.05) of respiratory rate than other treated nuts at the end of storage.
Free fatty acid content
showed the changes in free fatty acid content of pine nuts during storage. The experiments showed an increase in free fatty acid percentage with increasing storage time because of the degradation of large lipid molecules, which produced smaller molecules including free fatty acids by lipase and oxidation (Zhao, Xiong, Qiu, & Xu, Citation2007). The increase of free fatty acid content in high-moisture content nuts was higher than in the low-moisture content ones. Low-moisture treatment could reduce lipase activity. This might explain the beneficial effect of low-moisture treatment on storability of pine nuts. But free fatty acid content was not significantly affected when nuts were stored at −3°C or −1°C, the expected effect of temperature on free fatty acid formation was not observed on samples.
Figure 2. Changes in free fatty acid content and peroxide value of pine nuts during storage at −3 and −1°C: ▪, 17.3% moisture content (m.c.); ⧫, 15.1% m.c.; ▴, 13.3% m.c. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
Figura 2. Cambios en el contenido del ácido graso libre y en el valor de peróxido de piñones, almacenados a −3 y −1° C; ▪, 17.3% contenido de humedad (c.h.); ⧫, 15.1% c.h.; ▴, 13.3% c.h. Cada punto de datos es la media de tres muestras replicadas. Las barras verticales representan los errores estándar de las medias.
Peroxide value
Peroxide value is commonly used as an index of extent of oxidation and subsequently sensory rancidity. Oxidative extent of pine nuts as evaluated by peroxide value is summarized in . At the start of storage period, all samples exhibited low peroxide values, indicating that the pine nuts did not present the initial oxidative rancidity. Peroxide values continued to increase by the end of the storage, having a sharp rise from month 5 at −1°C. From month 3–12, it was observed that nuts with 13.3% ± 0.24 moisture content had significantly lower level of peroxide values compared to other moisture content nuts (p < 0.05). This suggested that lipid quality declined quickly as the moisture content increased, these were in agreement with the results obtained by Genkawaa et al. (Citation2008), who found that rice with decreased moisture content could lower fat acidity away from oxidation. From month 5–12, peroxide values of nuts with 13.3% ± 0.24 moisture content at −3°C were significantly lower than that at −1°C (p < 0.05). Similar results were obtained in 15.1% ± 0.26 moisture content and 17.3% ± 0.42 moisture content. Martin et al. (Citation2001) reported that low temperatures and the presence of shell effectively protected the hazelnut from oxidation. It was clear that storage at −3°C is more beneficial than −1°C for preservation of nuts. These clearly suggested that peroxide value was much related to moisture content and temperature, and low-moisture treatment and near freezing temperature storage could keep fresh, retard the oxidation and maintain low value in oxidative index of treated nuts.
Free amino acids content
Free amino acid contents from treated nuts are shown in . All treated nuts showed decreasing trend in free amino acid contents during the initial 4 months, then increased till to the end of storage. On month 6, there had been no significant change between all treatments, but on month 8, nuts with 13.3% ± 0.24 moisture content at −1°C and −3°C all demonstrated significantly lower level of free amino acids content when compared to other treatments (p < 0.05), but there was no significant difference between the two temperatures. At the end of storage, nuts with 13.3% ± 0.24 moisture content at −3°C showed significantly lowest level (p < 0.05) of free amino acids in all treatments. Free amino acids are responsible for special functions in primary and secondary metabolism of plants, e.g. seeds, as well as their role in protein synthesis and hydrolysis. Some free amino acids are used as a nitrogen source, whereas others are used as precursors of secondary products (Coruzzi & Last, 2000). The reason for free amino acids content initial declining was possibly relevant to lower respiration. In general, in the early storage protein hydrolysis occurs with a corresponding increase in free amino acids (Barker, Citation1987). We found that free amino acids content gradually raised between month 5 and 12, it implied an increase in proteinase activity, which accelerated the proteolysis.
Table 2. Free amino acids content (mg/100g) of pine nuts during 12 months storage.
Tabla 2. Contenido de aminoácidos libres (mg/100g) de piñones almacenados durante 12 meses.
The percentage of nuts with visible mold and browning
The percentage of visible mold observed in treated nuts was very little and could be neglected (). Until month 5, visible molds were not detected in all treated nuts. Nuts with 13.3% ± 0.24 moisture content were free from visible mold incidence after six months of storage. It was implied that low-moisture treatment delayed the appearance of visible mold growth during storage. Nuts with 17.3% ± 0.42 moisture content at −1°C had maximum visible mold percent (5.30%) occurring on month 12 of storage. Until the end of storage, percent of visible mold in 13.3% ± 0.24 moisture content nuts at −3°C was below 1%, followed by 13.3% ± 0.24 moisture content nuts (1.04%) at −1°C. It was similar to pecan nuts studied by Beuchat and Heaton (Citation2006), who suggested that pecan kernels at −6.5°C were less likely to support fungal growth than kernels at 0°C. Northolt and Bullerman (Citation1982) have reported that modified packaging with low-storage temperature prevented mold growth in ground nut. In the present study, it is suggested that integration conditions of low-moisture conditioned and near freezing temperature storage could strengthen the effect on reducing mold rate and keeping nuts in good quality. Changes in the browning of treated nuts () showed that the browning percent of treated nuts increased steadily with increased storage time, which was consistent with changes of nuts in peroxide value, indicating that oxidation extent of nuts increased gradually. Browning trend of nuts was similar to the visible mold percentage, and nuts in low-moisture content showed lower browning percent as compared to those in high-moisture content, while the browning percent of nuts stored at −3°C was a little lower than nuts stored at −1°C.
Table 3. Visible mold and browning percentage of pine nuts during 12 months storage.
Tabla 3. Porcentaje de moho visible y oscurecimiento de piñones almacenados durante 12 meses.
Sensory evaluation
Average values for the sensory attributes are shown in . The panel data indicated that until the month 3 the differences among treated samples in four sensory attributes were not significant (p > 0.05). There was no significant difference (p > 0.05) in color detected among all treated samples up to month 4, and at the end of storage, nuts with 13.3% ± 0.24 moisture content under two temperature had significant difference (p < 0.05) in color. Duncan's multiple range analysis showed that the differences among treated samples arose gradually in color, odor, texture, and taste with increased storage time. Until the end of storage, nuts with 17.3% ± 0.42 moisture content at −1°C had a color score of 6.0, odor score of 5.3 and taste near to 5.9. However, nuts with 13.3% ± 0.24 moisture content at −3°C had a color score on month 12 comparable to the score in noted near nuts on month 6. Similar trends were observed for odor and taste scores. Until the end of storage, nuts with 13.3% ± 0.24 moisture content under two temperatures had significant difference (p < 0.05) in odor and taste, and nuts at −3°C was better than at −1°C. These results support our previous findings and discussions of PV and free fatty acid. Hao, Heaton, and Beuchat (Citation1989) reported that low-temperature storage would maintain sensory quality of pecan packaged in hermetically sealed under ambient air in containers for long time. Nuts with 13.3% ± 0.24 moisture content at −3°C demonstrated a negative effect on texture which exhibited more crumbly, but they showed the best quality on other sensory attributes. According to the sensory analysis results, it could be summarized that nuts stored near freezing temperature integrated with low-moisture treatment remained overall sensory quality up to 12 months of storage.
Table 4. Changes of Sensory scores in pine nuts during 12 months storage.
Tabla 4. Cambios en la puntuación de aspectos sensoriales en piñones almacenados durante 12 meses.
Conclusions
It can be concluded from this study that 13.3% ± 0.24 moisture content and −3°C were most effective on maintaining high quality and delaying oxidative rancidity of pine nuts during long-term storage. Firstly, nuts with 13.3% ± 0.24 moisture content stored at −3°C kept lower respiration, free fatty acid content and peroxide value. Furthermore, they showed lower visible mold and browning percentage during storage. Though this combination of storage conditions had a negative effect on texture during storage, it had the best preservative effects on sensory quality of color, odor, and taste. In summary, low-moisture conditioning integrated with near freezing temperature storage effectively preserved the quality and extended the shelf-life of pine nuts.
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