Effect of Cold Storage on Saffron Flowers Shelf Life and Dried Stigma Quality

J. Appl. Environ. Biol. Sci., 4(12S)14-19, 2015 2015, TextRoad Publication ISSN: 2090-4274 Journal of Applied Environmental and Biological Sciences www.textroad.com Effect of Cold Storage on Saffron Flowers Shelf Life and Dried Stigma Quality Elham Azarpaahooh, Parvin Sharayei 1,2 1,2 Department of Food Science, Agricultural and Natural Resources Research Centre, Mashhad, Iran Received: November 21, 2014 Accepted: January 25, 2015 ABSTRACT Saffron (Crocus sativus L.) is a valuable crop in Iran and worldwide. Due to a rising production rate of saffron flowers and limitation of harvesting time, the best storage conditions should be determined. In this research, saffron flowers were harvested at semi-bloom form stage, and placed in plastic baskets, in two different thickness accumulations (10 and 15 cm). The flowers were stored at 0, 4, 8 and 21 C for after 2, 4, 7, 14 and 21 days. During storage period, weight loss percentage was calculated. In addition, the stigmas were separated from the styles and dried in an oven at 60 o C. Bioactive compounds such as crocin, picrocrocin and safranal, and coliform enumeration were evaluated. The result indicated that weight loss percentage increased with increasing storage thickness, temperature and storage duration; however bioactive compounds reduced. The crocin and picrocrocin content were registered 298.73 and 100, respectively, when flowers were stored at 0 o C for 21 days. These values at 8 o C and 21 o C were not in the standard range; therefore these temperatures were not found suitable for flower storing. Although coliform contamination increased during storage days, the value was acceptable after 7 days. According to the results, it is recommended to store saffron flowers in basket with 10 cm Thickness Accumulation (TA) at 0 o C for 7 days. KEY WORDS: Saffron flower, Storage, Temperature, Thickness accumulation INTRODUCTION Saffron is obtained from the dried stigmas of Crocus sativus L., which is a member of the Iridaceae family (Acar, Sadikoglu et al., 2011). Iran is considered to be the main producer of dried saffron all over the word with a production of 190 tons per year of dried saffron which is approximately 90% of the world s production (Bolandi, Shahidi et al., 2008, Gazor and Chaji 2010). It has been estimated that over 900,000 flowers and one hectare area are needed for producing 6 kg of saffron, (Souret and Weathers 2000). Considering that saffron corms require hand-planting, hand-picking, and hand-separating, stigmas from flowers make saffron production an extremely time-consuming and labor-intensive cultivation process (Winterhalter and Straubinger 2000; Gresta, Lombardo et al., 2008). Saffron flowering takes place in autumn, lasts only 2 3 weeks, and picking of the flowers is required almost daily as the flowers wilt rapidly and once this has happened they cannot readily be separated into their constituent parts (Basker 1999). Post harvest data in the literature largely refer to Iridaceae cut flowers, with little or no data on cold storage of cut saffron flowers in the world. Cut flowers have a very limited life after they have been cut off from the mother plant, as survival on their own reserves is generally low due to the special morphological and physiological characteristics of their tissues. The use of refrigeration for storage of flower cuttings is very important because it reduces water loss, senescence, infections caused by bacteria and fungi, thus extending the shelf- life of flowers during the storage period (da Silva Vieira et al., 2012). When harvested; cut flowers have a temperature of 5 C higher than the field ambient temperature. The correct procedure would be to remove as soon as possible that temperature. One of the techniques used for the removal of the "field heat" is to subject the flowers to the cooling process, also known as "pre -cooling", widely used for fruit and vegetables (Pellegrini and Belle, 2008, da Silva Vieira et al., 2012). The application of low temperature during storage is important for the conservation of the flowers, because in addition to inhibiting bacterial and fungal infections, it reduces degradation of certain enzymes and ethylene production; it reduces sweating, breathing and slows the various processes related to growth and senescence (Ahmad, Tahir et al., 2013). Temperature is considered the most important factor affecting the quality and longevity of cut flowers (Cevallos and Reid 2001, Turhan, Kahriman et al., 2010). At lower temperatures, flowers have a lower respiration rate and consume their stored energy much slower. Under physiological temperatures, a negative correlation has been observed between the increase in temperature and reduction of flower longevity (Cevallos and Reid 2001). Postharvest losses on account of prolonged separation of pistil, drying and packing have been a cause of low spot price due to poor quality on account of high microbial load (Nehvi, Wani et al., 2006). Cold storage often Corresponding author: Elham Azarpazhooh, Assistant professor, Khorasan Agricultural and Natural Resources Research Center, Mashhad, Iran. Email: elham.azarpazhooh@mail.mcgill.ca 14

Azarpaahooh and Sharayei, 2015 drastically increases the number of flower buds that do not open. Cold storage was also found to be a cause of petal wilting and aggravate leaf yellowing (van Doorn and Han 2011). Petal wilting is correlated with a decrease in petal free carbohydrate levels. It can be delayed by preventing the buds in the cut in florescence to act as a sink for carbohydrates. Depending on the cultivar, petal wilting is apparently not or only slightly regulated by ethylene (van Doorn and Han 2011). In contrast to high light, high temperatures during production tend to reduce vase life (Halevy and Mayak 1981), likely by increasing respiration rates and preventing accumulation of carbohydrate reserves. However, temperature effects are not well defined. Some reports indicate that increasing temperatures reduced vase life only at temperatures above approximately 25 C (Halevy and Mayak, 1979). Possible interactions between temperature and other factors complicate understanding temperature effects on vase life. For example, under low temperatures, carbohydrate reserves may increase, and pigments may also increase with low temperatures (Halevy and Mayak 1981; Chalker-Scott, 1999), causing plants to use carbohydrate reserves for pigment biosynthesis. The present study was undertaken to study the effect of storage layer thickness, dry storage temperatures and storage duration on the qualitative and quantitative characteristics of saffron flowers and their stigmas in order to develop an efficient and cost-effective storage protocol for this important spice. MATERIALS AND METHODS Sample Preparation The samples were collected from a 5 year-old saffron farm, situated 10 km far from Ghaen, South Khorasan Province, Iran. The flowers were manually picked daily early in the morning in the first hours after sunrise, before the flowers had completely opened (at the semi-open flower form stage). The flowers were completely mixed together and divided into 4 equal portions. The flowers were transferred to Toos Cold Store Factory near Mashhad, and stored in special cold rooms for further research purposes. The flowers were stored in baskets with two different thickness accumulations (10 and 15 cm) and stored at 0, 4, 8, and 21 o C, separately. The relative humidity in all storage cabinets was the same and equal to 95 ± 0.5%. The samples were bright out from cold store after 2, 4, 7, 14 and 21 days. It should be note that the stigmas were separated from the styles, and oven dried in a 60 C for 15 minutes. Physical and chemical analysis Weight loss Saffron flowers were weighed before and after the storage period to calculate weight Loss percentage. Bioactive Compounds Dried stigma samples were extracted in water. The absorbance was measured at 200 to 700 nm using an American spectrophotometer Model Genesys IM8. The crocin, picrocrocin and safranal content were determined according to the ISO 3632-2 method (ISO, 1993) and were computed by the following equation (1). A 10000 Amount = (1) 0.5( 100 H ) Where, A is absorbance value at 440, 257 and 330 nm for crocin, picrocrocin and safranal, respectively, on dry basis and H (%) is the moisture content of the stigmas (Saeidi Rad, et al., 2014)..Enumeration of microbes Microbial analysis of coliform was carried out on dried stigmas. Briefly, 10 g powdered saffron sample was isolated aseptically and diluted with 90 ml sterile peptone water (% 0.1) and blended for 10 min to prepare 1:10 dilution. Subsequent dilutions were prepared 1:100 and 1:1000. For coliform counts 1 ml of each dilution was spread onto plates containing Eosin methylene blue agar (Merck ) with two replicates and incubated for 48 h at 37 o C, in addition, the maximum possibility number (MPN) of coliform was obtained by the 3 tubes in lactose broth media for 24 h at 37 o C and 1 ml of each tube, which was positive for gas production, was added to another tube containing brilliant green broth (Merck ). Statistical analysis The experiments were carried out in a fully randomized factorial experimental design with three replications. MINITAB statistical analysis software was used for analysis of variance, ANOVA and Duncan s multiple range tests was utilized to separate means at a 5% level of significance. RESULTS AND DISCUSSION The weight loss percentage is shown in Fig 1. The results indicated that the weight loss percentage increased with increasing TA and temperature. As can be seen from Fig 1, the weight loss percentage was recorded less than 40% when the flowers were kept at 0 C with 15 cm TA for 7 days, whereas after 21 days, most of the flowers were still wet and the weight loss percentage was about 80%. Similarly, Joyce and Shorter 15

J. Appl. Environ. Biol. Sci., 4(12S)14-19, 2015 (2000) have reported that by increasing storage duration and temperature the respiration rate of the flowers would increase which make the flowers wilt. Tables 1, 2 and 3 are showing the influence of thickness accumulation and storage days on bioactive compounds of dried stigmas at different temperatures. The major bioactive compounds in saffron are crocin (mono-glycosyl or di-glycosylpolyene esters), picrocrocin (monoterpene glycoside precursor of safranal and product of zeaxanthin degradation), and safranal, all contributing not only to the sensory profile of saffron (color, taste, and aroma respectively) but also to the health-promoting properties (Rios, et al., 1996). The post-harvest processing such as storage conditions and temperature determine stability, quality, and economical value of the final product. The mean comparisons of the qualitative attributes are decreased by increasing the temperature and TA of the saffron flowers during storage. As can be seen from tables 1, 2 and 3, increase in temperature and storage days were caused to decrease crocin, picrocrocin and safranal.the highest qualitative characteristics were belonging to 0 C which showed a significant difference with stored samples at 21 C. In addition, during 7 days of storage, the qualitative characteristics were decreased significantly (p<0.05). The effect of TA and temperature on crocin content was also significant (p<0.05). The flowers, which were stored at 0 o C and TA of 10 cm showed the highest crocin content. The same results were observed in case of picrocrocin. The crocin and picrocrocin content were 320.1 and 108.03 at 0 o C, respectively; however these values were not in the standard range at 8 and 21 o C, therefore these temperatures were not found suitable for flower storing. The effect of TA and storage duration on coliform enumeration of dried stigmas at different temperatures is shown in table 4. The amount of coliform at 0 o C and TA of 10 and 15 cm were 0.75 10 3 and 0.80 10 3, respectively which reached to 2.49 10 3 and 5.62 10 3, at 21 o C. After 4 days, the amount of coliform increased, however this change was not statistically significant (p<0.05). After 7 days, significant differences were found in coliform enumeration, but these values were in the standard range. The comparison between the thicknesses showed that the 10 cm TA and 0 o C storage temperature had the lowest coliform contamination. Conclusion Since saffron plays an important role in economic growth and social life in Iran, performing any research that improves the quantitative and qualitative characteristics of this crop could directly affect the improvement of the farmers situation. The present study showed that thickness accumulation, temperatures and storage duration have significant effect on weight loss and bioactive compounds of saffron flowers. Based on the present research, the most suitable conditions for storing saffron flowers is 10 cm thickness accumulation, 0 o C and 7 days storing period. Acknowledgment We thank Khorasan Razavi Agriculture and Natural Resource Research Center for valuable support of our research programs. Table 1. The effect of TA and storage duration on crocin (λ max = 440 nm) content in dried stigmas stored at different temperatures (dried stigmas). Thickness Storage 0 o C 4 o C 8 o C 21 o C accumulation days 10 0 320.1±1.22a* 320.43±1.25a 321.78±0.34a 320.1±0.24a 2 318.31±0.25ab 309.61±0.86b 297.41±0.69b 269.95±1.53b 4 317.81±0.05b 284.86±0.56d 280.45±0.57c 260.27±0.52c 7 313.91±.0.7c 279.67±0.38e 271.93±0.41d 259.32±0.15c 14 303.66±0.08d 278.39±1.25e 267.33±1.05e 258.69±0.17c 21 298.73±0.03e 268.72±0.78 260.37±0.4g 253.58±0.76d 15 0 320.6±0.23a 320.1±0.76a 320.47±0.92a 321.27±0.04a 2 297.59±0.56e 300.31±1.45c 278.5±0.16c 270.24±0.11b 4 284.26±0.58f 280.81±0.78e 265.3±0.16ef 259.33±.0.27c 7 282.51±0.28g 279.55±1.04e 263.2±0.74f 255.09±1.9d 14 279.53±0.98h 270.62±0.09f 259.34±0.36g 253.09±1.3d 21 276.01±1.07I 265.73±1.04g 255.62±0.28h 248.35±0.4e *In each column, mean that at least one letter in common are significantly different according to Duncan s test at 5% level. 16

Azarpaahooh and Sharayei, 2015 Table 2. The effect of TA and storage duration on safranal (λ max = 330 nm) content in dried stigmas stored at different temperatures. Thickness Storage 0 o C 4 o C 8 o C 21 o C accumulation days 10 0 49.31±0.3a 48.58±0.05a 48.11±0.2a 48.48±0.48a 2 46.81±0.35b 47.63±.0.01ab 45.73±0.47b 42.95±0.06b 4 46.02±0.31b 43.3±0.35d 45.42±0.76b 43.87±0.12b 7 42.85±0.46c 41.09±0.08e 44.47±1.3b 43.53±0.16b 14 42.2±1.59c 41.16±0.2e 42.32±1.9c 39.17±0.2cd 21 42.34±0.2c 43.06±0.38e 40.17±0.2cd 35.54±0.34f 15 0 49.03±0.25a 48.96±0.34a 48.48±0.12a 48.97±0.17a 2 45.65±0.06b 46.65±0.2b 44.73±1.11b 40.46±0.04c 4 44.87±0.12c 45.13±0.15c 44.45±0.37b 38.08±0.12d 7 43.68±0.96c 43.89±0.12d 41.3±1.38c 37.03±0.23e 14 42.33±0.02c 42.38±0.18de 40.16±0.06cd 35.84±0.27f 21 41.05±0.08c 40.07±0.22e 39.87±0.28d 31.38±1.3g *In each column, mean that at least one letter in common are significantly different according to Duncan s test at 5% level. Table 3. The effect of TA and storage duration on picrocrocin (λ max = 257 nm) content in dried stigmas stored at different temperatures. Thickness Storage 0 o C 4 o C 8 o C 21 o C accumulation days 10 0 109.03±0.7a 107.7±0.07a 108.61±0.56a 108.28±0.31a 2 106.64±0.18b 107.72±0.04a 105.99±0.77b 100.46±0.69c 4 106.61±0.10b 101.32±0.04cd 105.66±0.61b 104.31±0.57b 7 102.41±0.08de 99.36±0.92d 105.12±0.08b 103.46±0.41b 14 100.56±0.21ef 98.96±0.07d 103.5±0.62c 94.31±1.05e 21 100.01±0.56f 103.51±1.59c 103.340.25c 80.35±0.40h 15 0 107.070.30a 107.53±1.3a 107.67±1.24a 107.92±0.92a 2 105.46±1.37b 106.61±0.12a 105.41±0.25b 100.24±0.16c 4 106.45±1.58b 106.4±0.51a 102.29±0.08cd 97.64±0.58d 7 104.44±0.14c 105.58±0.83b 100.51±0.22de 90.35±0.23f 14 103.88±0.7cd 105.61±1.27b 98.59±0.81ef 88.46±0.70g 21 101.22±1.44ef 103.72±0.59c 97.37±0.93f 79.31±0.16h *In each column, mean that at least one letter in common are significantly different according to Duncan s test at 5% level 90 80 70 Weight loss (%) 60 50 40 30 20 10 0 0 2 4 7 14 21 Day 0 oc 4 oc 8oC 21 oc Fig. 1. Weight loss of saffron flowers during storage at different temperatures 17

J. Appl. Environ. Biol. Sci., 4(12S)14-19, 2015 a 6 5 Coliform (10 3 ) 4 3 2 1 0 oc 4 oc 8 oc 21 oc b 0 0 2 4 7 14 21 Day 12 10 Coliform (10 3 ) 8 6 4 2 0 oc 4 oc 8oC 21 oc 0 0 2 4 7 14 21 Day Fig. 2. Influence of TA (a: 10 cm TA, b: 15cm TA), storage duration and temperature on coliform enumeration (Up: 10 cm, Down: 15cm), REFERENCES Acar B, Sadikoglu H, Ozkaymak M. 2011. Freeze drying of saffron (Crocus sativus L.). Drying Technology 29(14), 1622-1627. http://dx.doi.org/ 10.1111/jfpp.12214 18

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