Carbohydrate Polymers 127 (2015) 176–181

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Effect of drying methods on physicochemical properties and antioxidant activities of wolfberry (Lycium barbarum) polysaccharide Qingsheng Zhao a,1 , Beitao Dong a,b,1 , Jinjin Chen a,b , Bing Zhao a,∗ , Xiaodong Wang a , Liwei Wang a , Shenghua Zha a,b,c , Yuchun Wang a , Jinhong Zhang d , Yuling Wang d a

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190 Beijing, PR China Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China c Beijing Tong Ren Tang Health Pharmaceutical Co., Ltd., Beijing 100085, PR China d Yinchuan Taifeng Biotechnology Co., Ltd., Yinchun 750200, PR China b

a r t i c l e

i n f o

Article history: Received 5 February 2015 Received in revised form 28 February 2015 Accepted 23 March 2015 Available online 27 March 2015 Keywords: Lycium barbarum L. Polysaccharide Drying process Antioxidant activity

a b s t r a c t In this study, an efficient drying process of Lycium barbarum L. polysaccharide (LBP) suitable for industrial production was developed and optimized. Three drying methods, including hot air drying (40–80 ◦ C), vacuum drying (40–60 ◦ C) and spray drying were test and compared. Hot air drying and vacuum drying cost long time and produced a brown product which needs further process due to the agglomeration or alveolation form. The condition of spray drying (without any excipient) was optimized by orthogonal experiment, which gave different optimum conditions based on LBP recovery rate (LBP solution concentration 1.06 g/mL, inlet air temperature 170 ◦ C, sample flow rate 15 mL/min and air speed 4.2 m3 /min) or LBP transparency (LBP solution concentration 1.04 g/mL, inlet air temperature 170 ◦ C, sample flow rate 20 mL/min and air speed 2.8 m3 /min). Pilot scale experiments showed preferable stability of LBP product quality and process parameters. Sample of spray drying (SD) had the highest scavenging free radical effects, the best appearance (LBP transparency), and uniform morphology with hollow sphere which are important properties for the reconstitution of the powder product. Considering the product appearance and product activity, the spray drying was selected to apply in industrial production. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Lycium barbarum L. is an important traditional Chinese medicinal and edible plant found in China, Europe and the areas around the Mediterranean. Its red fruits contain 18 kinds of amino acids, including 8 kinds of essential amino acids and 21 kinds of trace minerals (the main ones being zinc, iron, copper, calcium, germanium, selenium, and phosphorus) (Yin & Dang, 2008). It also contains some functional nature components, such as flavonoids, carotenoids (Wang, Chang, Inbaraj, & Chen, 2010; Wang et al., 2010b) and polysaccharides (Liu et al., 2014; Qiu et al., 2014). Polysaccharides are the main active compounds in the fruits of L. barbarum (Ke et al., 2011). Studies have shown that L. barbarum polysaccharide (LBP) has a large variety of bioactivities, such as protect against irradiation injury (Li, Zhou, & Li, 2007), antioxidant activity (Liang, Jin, & Liu, 2011), increase bone gene expression

∗ Corresponding author. Tel.: +86 010 82627059; fax: +86 010 62574372. E-mail address: [email protected] (B. Zhao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2015.03.041 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

(Zhu et al., 2010), hypoglycemic effect (Zhu et al., 2013) and antivirus (Wang et al., 2010a,b). The annual production of fresh L. barbarum fruits were over 200,000 t in China (Yin & Dang, 2008). However, there are a few of them used in further industrial process. LBP is the most abundant constitutes, however, efficient industrial production of L. barbarum polysaccharides has been an important issue due to their complex physico-chemical properties. In all production processes, drying technology has been a bottleneck. In addition, the factors affecting the functions of polysaccharides were not only its chemical structure but also its molar masses and solution viscosity (Tikhonov et al., 2006). Drying process was also closely connection with the molar masses and solution viscosity of LBP. Thus, it is essential to develop a simple and efficient drying technology for industrial production of high quality polysaccharides from L. barbarum fruits. Drying is a widely used technology in food processing and preserving. However, drying process may cause irreversible changes of the polysaccharides, affecting their original structure, physiological and pharmacological properties of these polymers (Femenia, Simal, Taberner, & Rosselló, 2007; Nep & Conway, 2011). Several techniques have been employed for the drying procedure of

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Fig. 1. The illustrative diagram of L. barbarum polysaccharide at 0, 5 and 10 s.

polysaccharides. Conventional hot air drying (HD) is the most frequently used drying method in food industry. However, significant quality changes of dried products may occur during HD. Vacuum drying (VD) is ideal for materials which would be damaged or changed if exposed to high temperatures and oxygen. The vacuum removes moisture while preventing the oxidation that can occur when certain materials combine with air (Fan, Li, Deng, & Ai, 2012). However, the product in vacuum oven is easily bubbling which produce non-uniform drying products. The spray-drying (SD) process is ideal for polysaccharides which can produce a good quality product with low water activity and reduce the weight and facilitate easy storage and transportation (Amrutha, Hebbar, Prapulla, & Raghavarao, 2014; Murugesan & Orsat, 2012). This process is cost efficiency, operation with a high production rate of solids, better nutritional characteristics of the product due to the short contact time with high temperature (Marques et al., 2014). However, powders obtained by spray drying may have some problems in their properties, such as stickiness, hygroscopicity and solubility due to the presence of low-molecular-weight sugars and acids, which exhibit low glass transition temperatures (Bhandari, Senoussi, Dumoulin, & Lebert, 1993). Thus, one primary task of spray drying is to improve the adherence phenomenon of product to the walls of the drying chamber. The aim of the present study was to investigate the influences of different drying methods (HD, VD and SD) on the LBP and optimize the drying process to find the potential drying method suitable for the industrial production of bioactive L. barbarum polysaccharides. To the best of our knowledge, there were no reports on systematic studies of drying process of LBP.

Table 1 The drying time and appearance of the LBP at different temperature with different thickness of LBP paste when HD method was used. Temperature (◦ C)

Thickness of LBP paste (cm)

Drying time (day)

Appearance

40 ◦ C

0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5

2.5 5 7 3.5 4 5 2.5 3 4.5 2.5 3 3 1.5 2 2.5

Pale brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration Brown, agglomeration

50 ◦ C

60 ◦ C

70 ◦ C

80 ◦ C

2. Materials and methods 2.1. Materials Fresh L. barbarum specimens were provided by Yinchuan Taifeng Biotechnology Co., Ltd. and produced from Ningxia province, China. The materials were air-dried at room temperature. All other chemicals and solvents used were of analytical grade. 2.2. Extraction of LPB Polysaccharides from L. barbarum were prepared by the method of Zhao et al. (2011). The dried fruit samples were ground to fine powder and the polysaccharides product was extracted in ultrasonic circulating extraction equipment (CTXNW-2B, Beijing Hong Xiang Long Co., Ltd.). After the extraction, the mixtures were centrifuged at 5000 rpm/min for 15 min. The supernatant was concentrated by a rotary evaporator under reduced pressure to a proper volume and precipitated using 4 times volume 95% ethanol. After stirring, the precipitate was desired L. barbarum polysaccharide paste. 2.3. Viscosity of LBP paste The viscosity of the polysaccharide paste at different temperature was determined using a NDJ-1B viscometer (Changji Geological Instrument Co., Ltd). Table 2 The drying time and appearance of the LBP at different temperature with different thickness of LBP paste when VD method was used. Temperature (◦ C)

Thickness of LBP paste (cm)

Drying process

Drying time (day)

Appearance

40 ◦ C

0.5

Bubbling slightly Bubbling slightly Bubbling slightly Bubbling severely Bubbling severely Bubbling severely Bubbling severely Bubbling severely Bubbling severely

2

Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation Pale brown, alveolation

1 1.5 50 ◦ C

0.5 1 1.5

60 ◦ C

0.5 1 1.5

3.5 5 3 3.5 5 1.5 2 3

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Table 3 Recovery rate and transparency results of LBP in spray drying experiment on LBP. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Factors

Recovery rate (%)

(A) LBP solution concentration (g/mL)

(B) Inlet air temperature (◦ C)

(C) Sample flow rate (mL/min)

(D) Air speed (m3 /min)

1 (1.02) 1 1 1 2 (1.04) 2 2 2 3 (1.06) 3 3 3 4 (1.08) 4 4 4

1 (160 ◦ C) 2 (170 ◦ C) 3 (180 ◦ C) 4 (190 ◦ C) 1 2 3 4 1 2 3 4 1 2 3 4

1 (10) 2 (15) 3 (20) 4 (25) 2 1 4 3 3 4 1 2 4 3 2 1

1 (1.4) 2 (2.8) 3 (4.8) 4 (5.6) 3 4 1 2 4 3 2 1 2 1 4 3

36.32 90.00 79.94 73.74 87.16 90.58 32.32 70.97 80.65 67.81 94.84 58.58 45.81 51.61 62.26 76.13

Transparency at 1% 623 nm OD

0.767 0.652 0.646 0.676 0.403 0.452 0.485 0.420 0.620 0.651 0.697 0.882 0.828 0.673 1.759 1.432

2.4. Hot air drying procedure

2.8. Analysis of monosaccharide composition and FT-IR

Drying experiments of LBP pastes with different thickness (0.5, 1.0 and 1.5 cm) were carried out using HD at different temperatures (40 ◦ C, 50 ◦ C, 60 ◦ C, 70 ◦ C and 80 ◦ C) until the weight of LBP pastes became constant. The hot air drying oven (DGG-9140A) was made by Shanghai Shenxin Experimental Instrument Co., Ltd.

Polysaccharide sample (5 mg) was dissolved in 4 mL 2 mol/L trifluoroacetic acid solution (TFA) and hydrolyzed at 110 ◦ C for 3 h. The resulting solution was concentrated under reduced pressure and the excess acid was removed by repeated co-distillations with methanol. The monosaccharides were analyzed by gas chromatography. FT-IR of polysaccharides was carried out by the potassium bromide (KBr) pellet method on Fourier Transform-Infrared Spectrometer (FT/IR-660 Plus, JASCO) in the range of 400–4000 cm−1 .

2.5. Vacuum drying procedure VD experiments of LBP pastes with different thickness of LBP (0.5, 1.0 and 1.5 cm) were carried out at different temperatures (40 ◦ C, 50 ◦ C and 60 ◦ C) until the weight of LBP pastes became constant. The vacuum drying oven (DZ-1BC) was made by Tianjin Teste Instrument Co., Ltd. 2.6. Spray drying procedure Different concentrations of LBP solution was prepared for spray drying by dissolving LBP paste in deionized water without any excipient. An orthogonal L16 (4)4 test design was applied to investigate the optimal drying condition of LBP. LBP solution concentration (1.02, 1.04, 1.06 and 1.08 g/mL), inlet air temperature (160 ◦ C, 170 ◦ C, 180 ◦ C and 190 ◦ C), sample flow rate (10, 15, 20 and 25 mL/min) and air speed (1.4, 2.8, 4.2 and 5.6 m3 /min) were the 4 main factors of SD and the range of each factor was based on the results of preliminary single-factor experiments (date not shown). The recovery rate (%) and transparency of LBP were the dependent variables. The spray dryer (YC-015) was made by Shanghai Yacheng Experimental Instrument Co., Ltd. 2.7. Scanning electron microscopy The structure feature of LBP powder was observed by JSM-6700F scanning electron microscopy (SEM, JEOL, Japan). The sample was fixed on a metal stub with conductive tape, and was coated with platinum under vacuum by an ion sputter (JFC-1600, JEOL, Japan).

Table 4 Transparency results of LBP in three drying experiment. OD623

Hot air drying

Vacuum drying

Spray drying

1% OD623 0.5% OD623

2.735 1.772

1.992 1.218

0.362 0.135

2.9. Antioxidant activity assay The hydroxyl radical, superoxide radicals and DPPH scavenging activity of samples were measured according to the method of Yao et al. (2012). 2.10. Pilot spray drying test Optimal conditions of spray drying were used to verify the performance of SD in pilot scale with a LPG-5 spray dryer (5 L liquid sample per hour, Nanjing Kaiou Instrument Co., Ltd.). 2.11. Statistical analysis All experiments were performed at least in duplicate, and analyses of all samples were run in triplicate and averaged. Statistical analysis involved the use of the Statistical Analysis Systems (OriginPro 8.5) software package. The results were presented as means of three determinations ± SD (standard deviation). The results obtained were analyzed using one-way analysis of variance (ANOVA) for mean differences among the samples. P-Values of sample flow rate > LBP solution concentration > inlet air temperature. Under optimal spray drying condition LBP recovery rate was 96.13%. The spray drying orthogonal experiment results on LBP transparency are shown in Table 3. The optimal spray drying condition was as follow: LBP solution concentration 1.04 g/mL, inlet air temperature 170 ◦ C, sample flow rate 20 mL/min and air speed 2.8 m3 /min. The importance of influence factor was as follow order: LBP solution concentration > sample flow rate > inlet air temperature > air speed. Under optimal spray drying condition, LBP transparency was 0.376 at 1% in 623 nm and 0.135 at 0.5% in 623 nm which was significantly better than LBP drying by HD and VD (Table 4). Pilot scale experiments of SD showed preferable stability of LBP product quality and process parameters. No powder adhesions were found on the inner wall of the spray tower. Recovery rate of LBP powder reached more than 95%. Therefore, SD technology was suitable for industrial production of high quality LBP, which was stable, simple and low cost in drying process.

3.5. Scanning electron microscopy 3.4. The results of spray drying experiment result The spray drying method was a quick and effective drying technique for LBP. Considering the disadvantages of HD and VD, SD method properly solved the drying problem by increasing the specific surface area of sample and water evaporation rate. The LBP solution was dried quickly in a uniform micro-droplet. The viscosity was not more a problem in drying process of LBP. The product of SD LBP had high quality which not only in appearance but also the bioactivity. However the process parameters influenced the product quality of LBP significantly. Therefore, the optimal condition

The morphology of three LBP powders prepared by three drying technique was examined by SEM. As shown in Fig. 2, the LBP powder produced by SD that is smaller in size and was a more uniform size distribution than the HD or VD LBP samples. Particle morphology of the HD LBP sample is pyknotic and irregular. However, the HD LBP sample is loose. The more spherical shape and smoother surface texture of SD LBP powders have been attributed to the turbulences in the spray-drying process which cause some crush of the particle (the red arrow shows the fragment of SD LBP hollow sphere). The structure of LBP particles affected the

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wettability and solubility of LBP which was very important in LBP instant production development. 3.6. Antioxidant activity assay The antioxidant activity results of LBP are shown in Table 5. It can be seen that high temperature and long drying time significantly damage the LBP antioxidant activity. The DPPH, hydroxyl and superoxide free radical scavenging test have been widely accepted as tools for estimating the free-radical scavenging activities of antioxidants. The results indicated that LBP exhibited greater capacity in scavenging free radicals. Moreover, the antioxidant activity of polysaccharide dried by different process was influenced by the combination of various factors rather than one single factor. The scavenging abilities of HD and VD LBP were in a temperature-dependent fashion, higher drying temperature caused larger decrease of DPPH free radical scavenging ability. This phenomenon was also found in superoxide and hydroxyl radical scavenging effect. In addition, spray drying had worked advantages on preserving antioxidant activity because it can dehydrate the material very rapidly while providing some control of the particle size distribution, which meant little antioxidant activity damage during or after the drying process. The monosaccharides of LBPs by different drying methods with no difference were composed of rhamnose, arabinose, xylose, mannose, glucose and galactose in a ratio of 0.9:0.3:4.2:2.3:0.05:0.4. The FTIR spectra of LBPs by different drying methods are found to be similar, as is shown in Fig. 3. The band between 3260 and 3280 cm−1 , represents the stretching of the hydroxyl groups. The absorption bands around 2925 cm−1 are attributed to the C H stretching and bending vibrations. The peaks at around 1587 cm−1 are attributed to N H vibration or C O asymmetric vibration of carboxyl group. 4. Conclusion The results of the present work indicated that three polysaccharides obtained from the fruiting bodies of L. barbarum by different drying methods had different appearance and different levels of scavenging effects. Among three polysaccharides, LBP of SD had the highest scavenging effects, best appearance (LBP transparency) and a more uniform morphology, which was important properties for the reconstitution of the powder product. LBP of HD and VD exhibited satisfactory powder flow properties for application in tableting due to the aggregation state. In addition, we found that the viscosity of LBP was hugely influence the drying process and product quality. The SD technology successfully solved this problem without any carry agent. It’s enlightenment for the future study when the drying method must be selected by considering the sample characteristic and the product usage. Considering the all advantages, spray drying was the most suitable technology for industrial production of high quality of L. barbarum polysaccharides.

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Acknowledgements The authors gratefully acknowledge the financial support of the Yinchuan Taifeng Biotechnology Co., Ltd. for this Study. References Amrutha, N., Hebbar, H. U., Prapulla, S., & Raghavarao, K. (2014). Effect of additives on quality of spray-dried fructooligosaccharide powder. Drying Technology, 32(9), 1112–1118. Bhandari, B., Senoussi, A., Dumoulin, E., & Lebert, A. (1993). Spray drying of concentrated fruit juices. Drying Technology, 11(5), 1081–1092. Fan, L., Li, J., Deng, K., & Ai, L. (2012). Effects of drying methods on the antioxidant activities of polysaccharides extracted from Ganoderma lucidum. Carbohydrate Polymers, 87(2), 1849–1854. Femenia, A., Simal, S., Taberner, C. G., & Rosselló, C. (2007). Effects of heat treatment and dehydration on pineapple (Ananas comosus L Merr) cell walls. International Journal of Food Engineering, 3(2) http://dx.doi.org/10.2202/1556-3758.1097 Ke, M., Zhang, X.-J., Han, Z.-H., Yu, H.-Y., Lin, Y., Zhang, W.-G., et al. (2011). Extraction, purification of Lycium barbarum polysaccharides and bioactivity of purified fraction. Carbohydrate Polymers, 86(1), 136–141. Li, X., Zhou, A., & Li, X. (2007). Inhibition of Lycium barbarum polysaccharides and Ganoderma lucidum polysaccharides against oxidative injury induced by ␥irradiation in rat liver mitochondria. Carbohydrate Polymers, 69(1), 172–178. Liang, B., Jin, M., & Liu, H. (2011). Water-soluble polysaccharide from dried Lycium barbarum fruits: Isolation, structural features and antioxidant activity. Carbohydrate Polymers, 83(4), 1947–1951. Liu, Y., Gong, G., Zhang, J., Jia, S., Li, F., Wang, Y., et al. (2014). Response surface optimization of ultrasound-assisted enzymatic extraction polysaccharides from Lycium barbarum. Carbohydrate Polymers, 110, 278–284. Marques, G. R., Borges, S. V., Botrel, D. A., Costa, J. M. G. d., Silva, E. K., & Corrêa, J. L. G. (2014). Spray drying of green corn pulp. Drying Technology, 32(7), 861–868. Murugesan, R., & Orsat, V. (2012). Spray drying for the production of nutraceutical ingredients—A review. Food and Bioprocess Technology, 5(1), 3–14. Nep, E. I., & Conway, B. R. (2011). Physicochemical characterization of grewia polysaccharide gum: Effect of drying method. Carbohydrate Polymers, 84(1), 446–453. Qiu, S., Chen, J., Chen, X., Fan, Q., Zhang, C., Wang, D., et al. (2014). Optimization of selenylation conditions for Lycium barbarum polysaccharide based on antioxidant activity. Carbohydrate Polymers, 103, 148–153. Tikhonov, V. E., Stepnova, E. A., Babak, V. G., Yamskov, I. A., Palma-Guerrero, J., Jansson, H.-B., et al. (2006). Bactericidal and antifungal activities of a low molecular weight chitosan and its N-2 (3)-(dodec-2-enyl) succinoyl-derivatives. Carbohydrate Polymers, 64(1), 66–72. Wang, C., Chang, S., Inbaraj, B. S., & Chen, B. (2010). Isolation of carotenoids, flavonoids and polysaccharides from Lycium barbarum L. and evaluation of antioxidant activity. Food Chemistry, 120(1), 184–192. Wang, J., Hu, Y., Wang, D., Zhang, F., Zhao, X., Abula, S., et al. (2010). Lycium barbarum polysaccharide inhibits the infectivity of Newcastle disease virus to chicken embryo fibroblast. International Journal of Biological Macromolecules, 46(2), 212–216. Yao, L., Zhao, Q., Xiao, J., Sun, J., Yuan, X., Zhao, B., et al. (2012). Composition and antioxidant activity of the polysaccharides from cultivated Saussurea involucrata. International Journal of Biological Macromolecules, 50(3), 849–853. Yin, G., & Dang, Y. (2008). Optimization of extraction technology of the Lycium barbarum polysaccharides by Box–Behnken statistical design. Carbohydrate Polymers, 74(3), 603–610. Zhao, Q., Kennedy, J. F., Wang, X., Yuan, X., Zhao, B., Peng, Y., et al. (2011). Optimization of ultrasonic circulating extraction of polysaccharides from Asparagus officinalis using response surface methodology. International Journal of Biological Macromolecules, 49(2), 181–187. Zhu, J., Liu, W., Yu, J., Zou, S., Wang, J., Yao, W., et al. (2013). Characterization and hypoglycemic effect of a polysaccharide extracted from the fruit of Lycium barbarum L. Carbohydrate Polymers, 98(1), 8–16. Zhu, M., Jinggang, M., ChangSheng, H., Haiping, X., Ning, M., & Caijiao, W. (2010). Extraction, characterization of polysaccharides from Lycium barbarum and its effect on bone gene expression in rats. Carbohydrate Polymers, 80(3), 672–676.