Astaxanthin from Phaffia rhodozyma: Microencapsulation with carboxymethyl cellulose sodium and microcrystalline cellulose and effects of microencapsulated astaxanthin on yogurt properties

Accepted Manuscript Astaxanthin from Phaffia rhodozyma: Microencapsulation with carboxymethyl cellulose sodium and microcrystalline cellulose and effects of microencapsulated astaxanthin on yogurt properties Zhao-Zhao Feng, Ming-Yuan Li, Yu-Tao Wang, Ming-Jun Zhu PII:

S0023-6438(18)30399-2

DOI:

10.1016/j.lwt.2018.04.084

Reference:

YFSTL 7095

To appear in:

LWT - Food Science and Technology

Received Date: 8 November 2017 Revised Date:

26 April 2018

Accepted Date: 27 April 2018

Please cite this article as: Feng, Z.-Z., Li, M.-Y., Wang, Y.-T., Zhu, M.-J., Astaxanthin from Phaffia rhodozyma: Microencapsulation with carboxymethyl cellulose sodium and microcrystalline cellulose and effects of microencapsulated astaxanthin on yogurt properties, LWT - Food Science and Technology (2018), doi: 10.1016/j.lwt.2018.04.084. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Astaxanthin from Phaffia rhodozyma： ：

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microencapsulation with carboxymethyl cellulose sodium and microcrystalline cellulose and

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effects of microencapsulated astaxanthin on yogurt properties

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Zhao-Zhao Feng a, Ming-Yuan Li b, c, Yu-Tao Wang b, c, Ming-Jun Zhu *a, b, c

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a

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Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of

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China

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b

College of Life and Geographic Sciences, Kashgar University, Kashgar 844000, China

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c

The Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges &

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School of Biology and Biological Engineering, South China University of Technology,

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Universities under the Department of Education of Xinjiang Uygur Autonomous Region, Kashgar

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University, Kashgar 844000, China

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* Corresponding author; E-mail address: [email protected]; Tel: +8620 39380623

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Abstract In this study, the astaxanthin from Phaffia rhodozyma was encapsulated with carboxymethyl

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cellulose sodium (CMC-Na) and microcrystalline cellulose (MCC) by freeze-drying and used in

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yogurt. The encapsulation improved the stability, solubility, and antioxidation activity of the

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astaxanthin and increased the extent of its potential industrial application. The encapsulated

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efficiency and solubility of the microencapsulated astaxanthin were 58.76% and 52.88%,

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respectively. In comparison with non-encapsulated astaxanthin, the microencapsulation did

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prevent the astaxanthin from dramatic degradation, with an astaxanthin retention value of 80.86%

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in the microencapsulated astaxanthin at 55

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much more stable in acid than in neutral phase, but the microencapsulated astaxanthin showed no

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significant difference in stability under both conditions. The astaxanthin yogurt showed an

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attractive orange-red color as well as a significantly higher DPPH-scavenging activity and stability

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than the plain yogurt.

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Keywords: microencapsulation; astaxanthin; carboxymethyl cellulose sodium; microcrystalline

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cellulose; yogurt

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in 16 hours. Non-encapsulated astaxanthin was

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1. Introduction Astaxanthin, one of the carotenoid pigments, is widely distributed in nature, especially in

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shrimp, crab, fish, algae, birds, yeast and Haematococcus pluvislis. Astaxanthin is well known for

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its health-promoting properties and stain ability. Its bioactivity is higher than that of any other

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known antioxidants and it could protect lipids of biological membranes from peroxidation

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(Preston et al., 2006). Inflammatory and oxidative mediated disorders including cancer, allergy,

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diabetes, neurodegenerative diseases and coronary heart diseases could be reduced by its

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polyunsaturated fatty acids (Anarjan, Tan, Nehdi & Ling, 2012; Bustos-Garza, Yanez-Fernandez,

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& Barragan-Huerta, 2013). Astaxanthin could be favorable to animal and human health probably

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due to its ability to eliminate free radical and singlet oxygen (Gomez-Estaca, Comunian, Montero,

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Ferro-Furtado, & Favaro-Trindade, 2016). Furthermore, astaxanthin could also induce

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NF-E2-related factor 2 (Nrf2), which has been experimentally found to prevent cells from

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oxidative stress damage (Inoue et al., 2017). Oral astaxanthin prodrug CDX-085 could distribute

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among lipoproteins and lower total cholesterol and aortic arch atherosclerosis in low-density

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lipoprotein receptor negative mice (Ryu et al., 2012). Researchers demonstrated that feeding

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astaxanthin-supplemented diets could improve long snout seahorses’ egg quality and juvenile

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growth and survival (Palma, Andrade, & Bureau, 2017). Consequently, astaxanthin is widely used

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as a nutrition additive in various promising fields.

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Despite its steadily increasing need in recent years, astaxanthin is still stifled by its strong

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odor, low aqueous solubility, rapid metabolism, highly conjugated structure and unsaturated nature.

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Native astaxanthin is red in color but it turns into blue or purple when complexed with proteins or

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lipoproteins (Higuera-Ciapara, Felix-Valenzuela, Goycoolea, & Arguelles-Monal, 2004). Free

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astaxanthin is susceptible to light, temperature, pH, and oxygen during thermal treatment or

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storage

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Hernandez-Munoz, 2012). Thus, it is necessary to overcome these limitations to improve

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astaxanthin properties for potential industrial applications.

Mozafari,

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Mohebbi,

2012;

Gomez-Estaca,

Balaguer,

Gavara,

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(Fathi,

There are many ways to solve the astaxanthin limitations such as microencapsulation with

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emulsions, liposomes, polymeric nanospheres, β-cyclodextrin complexes or calcium ions (Ambati,

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Phang, Ravi, & Aswathanarayana, 2014 ; Raposo & Morais, 2012). Ai and Nyam (2016)

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fabricated a stable kenaf seed oil-in-water Pickering nanoemulsion by mixing sodium caseinate

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(SC), Tween 20 (T20) and β-Cylodextrin (β-CD) through a high pressure homogenizer. Tan, Xie,

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Zhang, Cai, and Xia (2016) developed the polysaccharide-based nanoparticles by the

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polyelectrolyte complex between chitosan (CS) and gum arabic (GA) as novel delivery systems

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for curcumin. Gomez-Estaca et al. (2016) used the novel biopolymer combination of gelatin and

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cashew gum to encapsulate an astaxanthin-containing lipid extract obtained from shrimp waste by

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complex coacervation. Microencapsulation of astaxanthin can be performed by emulsion/solvent

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evaporation, freeze-drying, solvent displacement and spray drying (Taksima, Limpawattana, &

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Klaypradit, 2015). The aforementioned studies suggest that the encapsulation technology to enable

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solid powders to contain the lipid extract might be a good way to tackle astaxanthin limitations. In

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this work CMC-Na and MCC are used as the wall materials of encapsulation on the study of the

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stability of astaxanthin from P. rhodozyma.

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CMC-Na, one of the important water-soluble cellulose ether derivatives with negative

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charges, is synthesized by the alkali-catalyzed reaction of cellulose with chloroacetic acid (Miao,

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Chen, Li, & Dong, 2007). CMC-Na is usually used as a water-soluble thickener, emulsifier and

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ACCEPTED MANUSCRIPT film-former in biomedical membrane and tablet coating (Salum et al., 2006). MCC could form a

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stable gel in water with a great number of special properties such as oil absorption, large surface

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area, non-toxicity, biodegradability, low density and biocompatibility, etc (Miao & Hamad, 2013;

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Yang, Tang, Wang, Kong, & Zhang, 2014; Zulkifli, Samat, & Anuar, 2015). It also exhibits a

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unique capacity to improve the morphology, thermal and mechanical properties of the composite

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(Xiao, Qi, Zeng, Yuan, & Yu, 2014). MCC is difficult to disintegrate once dried because the

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hydrogen bonds in MCC cause strong adhesion between the individual micro fibrils (Comunian,

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Thomazini, Alves, Balieiro, & Favaro-Trindade, 2013). So far, there is no report available on the

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use of the CMC-Na and MCC for astaxanthin encapsulation. It is useful to minimize the

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aforementioned disadvantages of free astaxanthin, which is favorable to be used in the food,

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cosmetology, and medicine industry.

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Natural bioactive compounds have been increasing focus with the development of society

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compared with synthetic chemicals. However, it is also important to choose a suitable food

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product for the encapsulated astaxanthin. Yogurt, a daily consumed healthy, nutritious and tasty

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dairy, is rich in vitamins, minerals, calcium and proteins (Panagiotidis & Tzia, 2001). More

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importantly, it can deliver probiotics to improve the health of consumers. However, yogurt is

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deficient in total antioxidant activity and stability (Krasaekoopt, Bhandari, & Deeth, 2006). It is

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necessary to develop new functional yogurt to meet people’s appetite and market requirements.

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Therefore, the study on the encapsulation of astaxanthin with CMC-Na/MCC to improve the total

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antioxidant activity of yogurt and its stability for longer shelf life is meaningful.

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In the present work, the stability and properties of astaxanthin from P. rhodozyma was

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investigated by encapsulation with MCC and sodium CMC-Na. Additionally, the effects of the

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microencapsulated astaxanthin on yogurt properties were also examined in terms of storage

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stability, color, DPPH-scavenging activity, total number of lactic acid bacteria and acidity.

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2. Materials and methods 2.1. Materials

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P. rhodozyma Y119 was stored in the Institute of Biological Sciences and Engineering of

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South China University of Technology, Guangzhou, China. CMC-Na was purchased from Tianjin

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Zhiyuan Chemical Reagent Factory (China). MCC was Avicel PH 105, FMC Corporation, USA.

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All other reagents were of analytical grade and acquired from Sigma-Aldrich, USA.

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2.2. Extraction of astaxanthin

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The cells of P. rhodozyma were harvested with a centrifuge at 5000 rpm for 3 min, then

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resuspended and washed twice with distilled water. The supernatant was completely removed after

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centrifugation. In the process of acid washing, 1 mL 3 mmol/L HCl was added into 1mL wet cells

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of P. rhodozyma and stood for 30 min, followed by boiling for about 4 min, then bathing in ice

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water immediately util cooling down and centrifugation at 5000 rpm for 3 min. After that, the

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supernatant was removed and the cells of P. rhodozyma were used for further solvent extraction

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(Yang & Ji, 1995).

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The extraction was performed at room temperature for 1 hour protected from light. The cell

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debris was removed by centrifugation at 5000 rpm for 5 min at 4 °C. The supernatant was

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collected to detect the concentration of astaxanthin using a spectrophotometer. Absorbance was

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measured at 474 nm. Total astaxanthin concentration was calculated using the following equation

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Eq. (1) (Gomez-Estaca et al., 2016):

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Carotenoid (mg ) = A 474 * V* P/ξ

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(1)

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where A is the absorbance at 474 nm; V, the dilution volume (mL); P and ε, the molecular

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weight and the molar absorption coefficient of astaxanthin (597 and 125,100, respectively).

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2.3. Preparation of inclusion complex MCC and CMC-Na powders were mixed at 3:7, 4:6, 5:5, 6:4, 7:3, with distilled water added

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under stirring for 30 min (CMC-Na: water =1:20). The suspension was homogenized by a high

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speed dispersing homogenizer with digital display system (FJ-200SH) at 18,000 rpm for 5 min.

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Suspension was transferred into plate containers and freeze-dried. Next, the freeze-dried samples

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were grinded into powders by blender and stored at 4 ℃ for further use.

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To measure the stability of the inclusion complex suspension, the powder was mixed with

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distilled water under stirring for 30 min (inclusion complex: water =1:3), followed by centrifuging

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1 mL inclusion complex suspension at 3000 rpm for 5 min, discarding the supernatant, and

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weighing the sediment accurately. The stability of the inclusion complex suspension was

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estimated by the following Eq. (2)(Yang & Zhao,1993);

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The stability of inclusion complex suspension (%) =

Sediment (g) * 100 Suspension (g)

(2)

2.4. Preparation of microencapsulated astaxanthin

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CMC-Na and MCC were dissolved in astaxanthin acetone extract solution (CMC-Na:

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MCC=6:4, acetone:CMC-Na =3:1), with distilled water added under stirring for 30 min (CMC-Na:

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water =1:20). The process was the same as described in section 2.3.

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2.5. Preparation of astaxanthin yogurt

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Two different kinds of yogurt adding astaxanthin or not in two groups were prepared: yogurt

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a (control), astaxanthin yogurt a, yogurt b (control), and astaxanthin yogurt b (a is sucrose yogurt

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and b is oligosaccharide yogurt). The amount of astaxanthin added was approximately 8 mg per

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ACCEPTED MANUSCRIPT 100 g yogurt, about 2% microencapsulated astaxanthin. The yogurt samples were stored at 4 ℃

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for further analysis.

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2.6. Analytical methods

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2.6.1. FT-IR

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Fourier transform infrared spectroscopic (FT-IR) spectra of the samples (CMC-Na, MCC,

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non-encapsulated astaxanthin, microencapsulated astaxanthin) were obtained in the range from

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500 to 4000 cm-1using a PerkinElmer Spectrum(Yang, Gao, Hu, Li, & Sun, 2015).

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2.6.2. Encapsulated efficiency

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Encapsulated efficiency (EE) was calculated on the basis of the total quantity of astaxanthin

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in the microencapsulated astaxanthin and non-encapsulated astaxanthin present on the surface

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(Bustamante, Masson, & Velasco, 2016). The surface astaxanthin was determined as follows: 100

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mg of freeze-dried microencapsulated astaxanthin was accurately weighed and washed twice with

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2 mL of acetone, with the two volumes of acetone mixed; and the total quantity of astaxanthin was

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determined as follows: 100 mg of freeze-dried microencapsulated astaxanthin was mixed with 1

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mL distilled water and 2 mL acetone, vortexed for 5 min, then recovered by extraction with 2 mL

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acetone. EE was calculated according to Eq. (3) (Daniels & Mittermaier, 1995):

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EE（%）=（

Total amount of astaxanthin - nonencapsulated astaxanthin ）* 100 Total amount of astaxanthin

(3)

2.6.3. Solubility

The solubility of the microencapsulated astaxanthin was determined using a gravimetric

method (Comunian et al., 2013).

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Briefly, 0.5 g microencapsulated astaxanthin was mixed with 50 mL distilled water in a

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conical flask at 22 ℃and 220 rpm for 30 min. The solution was then centrifuged at 1,400 g for 5

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min, and an aliquot of the supernatant was transferred to previously weighed bottles and

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maintained in an oven at 105

The solubility of the astaxanthin was calculated according to Eq. (4):

Solubility(%) =

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2.6.4. Thermal and pH stability study

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(4)

The effects of temperature and pH on the stability of microencapsulated astaxanthin and non-encapsulated astaxanthin were tested as previously reported (Jung, Kim, & Shin, 2005).

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Supernatant dried weight * 100 0.5

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until constant weight.

Briefly, aliquots of microencapsulated astaxanthin and non-encapsulated astaxanthin were

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accurately obtained, with pH adjusted to 1.5 and 6.8, and then maintained in an incubator at 55 ℃

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in darkness for a different period.

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Total astaxanthin content during storage was determined as described above. The retention rate (RR, %) was quantified according to Eq. (5):

RR（ %） =

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(5)

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The DPPH-scavenging activity of microencapsulated astaxanthin and yogurt was measured as previously reported (Taksima, Limpawattana, & Klaypradit, 2015).

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Total amount of astaxanthin after storage * 100 Total amount of astaxanthin initially prepared

2.6.5. DPPH-scavenging activity

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Briefly, 2 mL DPPH solution in alcohol (0.1 g /L) was added to 0.5 mL samples (yogurt was

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diluted 10 times), then shaken vigorously and allowed to stand in the dark at room temperature for

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30 min. The absorbance of the sample solution was measured at 580 nm using a

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spectrophotometer. The percentage of DPPH-scavenging activity was calculated according to Eq.

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(6):

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DPPH- scavengingactivity(%)=

Acontrol- (Asample- Asampleblank) * 100 Acontrol 9

(6)

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Acontrol, Asample, and Asample

blank

are the absorbance of the DPPH solution in alcohol,

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astaxanthin solution with DPPH, and astaxanthin solution without DPPH, respectively.

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2.6.6. Color measurement The colors of the microencapsulated astaxanthin and astaxanthin yogurt were determined

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using a portable colorimeter (Konica Minolta, CM-700D) as previously described (Anarjan, Tan,

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Nehdi, & Ling, 2012).

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2.6.7. Yogurt properties

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To evaluate the shelf life of different kinds of yogurt, equal samples were centrifuged at 3000

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rpm for 5 min, and the supernatant was discarded. The stability index was measured and

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calculated according to Eq. (7) (Surh et al., 2006):

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Storage stability index (%) =

Final weight * 100 Initial weight

(7)

Total number of lactic acid bacteria and acidity were measured by GB 4789.35-2016. Each

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analysis was performed in triplicate.

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3. Results and Discussion

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3.1. Preparation of inclusion complex

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The formation of the inclusion complex (without astaxanthin extract addition) was studied at

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a different ratio of MCC to CMC-Na from 3:7 to 7:3. At the studied pH (3.1 and 6.8), the ratio of

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3:7 or 4:6 (MCC: CMC-Na) was more stable than that of the other ratios (Table 1). Because MCC

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could form a stable gel in water with special oil absorption and low viscosity properties (Xiao et

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al., 2014), it was used in yogurt and the ratio of 4:6 (MCC: CMC-Na) was adopted for further

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study. Additionally, the inclusion complex remained stable from pH 3.5 to 10.8 in 5 days after test

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and was only slightly unstable at pH 2.8 (Table 2). All the data indicated the proportion of

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suspension (MCC: CMC-Na = 4:6) was appropriate.

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3.2. Characterization of microencapsulated astaxanthin The microencapsulated astaxanthin obtained by freeze-drying was investigated by optical

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microscopy (Fig.1C) and scanning electron microscopy (SEM) (Fig.1D & E & F). As can be seen

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from Fig.1C, the microencapsulated astaxanthin had an obvious red color. Compared with Fig.1A

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and B, MCC and CMC-Na tended to aggregate and form larger size clusters. The appearance of

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the CMC-Na was relatively smooth (Fig.1A), while resultant powders combined with CMC-Na

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and MCC had irregular morphology with deep dents and holes (Fig.1F), which resulted in a larger

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surface area. The depression and concave-convex on the surface of microencapsulated astaxanthin

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could adsorb more astaxanthin, protecting it from oxidation and improving its encapsulated

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efficiency.

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Fig.2 shows the FT-IR spectra of CMC-Na, MCC, microencapsulated astaxanthin and

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non-encapsulated astaxanthin. MCC exhibited a typical band at 400-500 cm-1(Fig.2C) while

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CMC-Na showed two strong bands at 950-1100 cm-1 and 500-600 cm-1 (Fig.2B). These

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characteristic peaks were also observed in the microencapsulated astaxanthin (Fig. 2A). In the case

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of astaxanthin, strong bands were detected at 950-1100 cm-1 and 1500-1650 cm-1 (Fig.2D). These

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characteristic peaks were attenuated in the microencapsulated astaxanthin. Under the interaction of

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wall materials and astaxanthin, the FT-IR spectrum of the microencapsulated astaxanthin changed

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greatly at 400-2000 cm-1, indicating a successful encapsulation.

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Table 3 shows the properties of the microencapsulated astaxanthin. The ultrasonic atomizer

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was reported to be successfully applied for astaxanthin encapsulation with alginate-chitosan,

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which had an average diameter of 4.23 mm (Taksima et al., 2015). The size of our

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ACCEPTED MANUSCRIPT microencapsulated astaxanthin had a smaller diameter about 0.074 mm. Astaxanthin was also

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encapsulated with gelatine-cashew gum by freeze-drying, which had an average size of 0.032 mm

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(Gomez-Estaca et al., 2016). However, gelatin is not dissolved in cold water, which may be

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unfavorable to the use of astaxanthin.

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The water solubility of the particles studied in the present work was 52.88 % (Table 3), which

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was much higher than that of the non-encapsulated astaxanthin (insoluble in water). It was also

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higher than that of the gelatine-cashew gum complex (28.60 %) (Gomez-Estaca et al., 2016). The

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microencapsulated astaxanthin had values of 5.22 and 7.17 for △a* and △b*, respectively, which

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is greater than 0, indicating that it exhibited a red-orange color (Table 3).

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Encapsulated efficiency (EE) can explain whether the core material is protected and the

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astaxanthin properties are improved. In the present study, the EE value of astaxanthin was 58.76%

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(Table 3), which was similar to the 59.90% of the gelatine-cashew gum complex (Gomez-Estaca

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et al., 2016). In several previous studies, the EE reached 90%, such as 85-91% for

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alginate-chitosan astaxanthin encapsulation (Taksima et al., 2015), and 90% for chitosan and gum

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arabic polyelectrolyte complex for curcumin. The EE in the present study might be further

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enhanced by improving the concentration of CMC-Na or pretreating wall materials, but it needs

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further research.

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These results confirmed the potential use of CMC-Na and MCC as astaxanthin carriers,

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demonstrating their effectiveness in increasing the solubility of astaxanthin by encapsulation.

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3.3. Thermal and pH stability

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As shown in Fig.3, astaxanthin encapsulation by freeze drying could avoid the heating loss of

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astaxanthin compared with spray drying. Thermal instability is a common problem in astaxanthin.

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An increased temperature always accelerated astaxanthin degradation. Heating and lighting in the

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presence of oxygen can dramatically lead to the isomerization and degradation of astaxanthin

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(Boon,

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Villanueva-Rodriguez, 2017) . The β-carotene in benzene or carbon tetrachloride in the presence of

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oxygen was completely lost within 30 hours in the dark at 30 ℃ (Ambati, Phang, Ravi, &

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Aswathanarayana, 2014). Experiments showed that the thermal stability of astaxanthin could be

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improved by using microencapsulation.

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The content of non-encapsulated astaxanthin decreased sharply during storage and the value

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of astaxanthin retention was 62.67% in 16 hours. In comparison with non-encapsulated

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astaxanthin, the microencapsulated astaxanthin did prevent itself from dramatic degradation, with

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an astaxanthin retention value of 80.86% under the same condition, and the degradation rate of

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astaxanthin in microencapsulated astaxanthin was obviously retarded (Fig. 3).

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Astaxanthin might induce carbocation formation when exposed in acid environment

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(Villalobos-Castillejos, Cerezal-Mezquita, Hernandez-De Jesus, & Barragan-Huerta, 2013).

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Studies found that the stability of the astaxanthin decreased with pH increasing from 4 to 7

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(Villalobos-Castillejos et al., 2013). Therefore, a pH simulation of gastric and intestinal organs

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(pH 1.5 and 6.8, respectively) to examine the astaxanthin stability was performed. In comparison

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with non-encapsulation astaxanthin, the microencapsulated astaxanthin prevented astaxanthin

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from degradation at 55 ℃. The effect of the studied pH values on stability was not significant for

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the microencapsulated astaxanthin, but significant for the non-encapsulated astaxanthin, which

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was less stable in neutral than in acid phase (Fig.4A & B).

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These results demonstrated the superiority of astaxanthin encapsulation by using CMC-Na

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ACCEPTED MANUSCRIPT and MCC. Gomez-Estaca et al. (2016) performed an accelerated stability study and found an

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improvement in astaxanthin stability after encapsulation with gelatine-cashew gum. Qian, Decker,

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Xiao, and McClements (2012) discovered astaxanthin degradation was slower in β-lactogloblin

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nanoemulsion at 55 ℃, but it was unstable at a lower pH. Comparatively, our microencapsulated

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astaxanthin was more stable in acid solution.

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3.4. DPPH-scavenging activity

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The DPPH-scavenging activity of astaxanthin was evaluated as antioxidation activity. The

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DPPH-scavenging activity of the microencapsulated astaxanthin was slightly changed over time at

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55 ℃, while that of non-encapsulated astaxanthin had a steady decline from 86.18% to 27.64%

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(Fig.4C). The results showed that astaxanthin had a great antioxidant function but extreme

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instability, suggesting that the encapsulation was helpful to avoid the release of astaxanthin and

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could efficiently protect it from oxidation. Due to the insolubility of CMC-Na and MCC in organic

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solvents, the value of DPPH-scavenging activity was lower, but the antioxidation activity was high

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and steady (Fig. 4C). A previous study also found the value of DPPH-scavenging activity of

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astaxanthin complex with hydroxypropyl-cyclodextrin became lower due to the hindrance to the

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reaction between astaxanthin and hydroxyl radical (Yuan, Du, Jin & Xu, 2013).

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3.5. Effects of the microencapsulated astaxanthin on yogurt properties

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The microencapsulated astaxanthin was added into yogurt as a food thickener, stabilizer and

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coloring additive, which was homogeneously dispersed in yogurt. The astaxanthin yogurt samples

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showed an attractive orange-red color and 9.41% higher stability compared with the control

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samples (Table 4). Non-encapsulated astaxanthin was hydrophobic, and it was better dispersed

294

when encapsulated. In previous studies, the yogurt treated with the astaxanthin encapsulated by

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ACCEPTED MANUSCRIPT alginate-chitosan had an overall liking score of over 6.0 (on the 9-point scale), a positive

296

acceptance of 86.2% and a purchase intent 95.6% (Taksima et al., 2015), and the yogurt with

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added astaxanthin encapsulated by gelatine-cashew gum had a more intense odor than the plain

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one because of low encapsulated efficiency (Gomez-Estaca et al., 2016). In the present study, the

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astaxanthin yogurt had a normal flavor.

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The DPPH-scavenging activity in yogurt was identified to examine the antioxidation activity

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of the astaxanthin yogurt. As shown in Fig. 5A and B, the DPPH-scavenging activity of the

302

astaxanthin yogurt was significantly higher than that of the control and could maintain longer

303

activity. The use of microencapsulated astaxanthin as a functional yogurt supplement maximized

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its characteristics, such as appropriate pH, temperature and low oxygen content. Meanwhile, the

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microencapsulated astaxanthin enhanced the yogurt stability and antioxidation activity. These

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results showed the great potential of the astaxanthin yogurt in the future yogurt market.

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As shown in Fig.5C-F, the total number of lactic acid bacteria in the astaxanthin yogurt had

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no significant difference from that of the plain yogurt, and the acidity of the astaxanthin yogurt

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was slightly higher than that of the plain one during fermentation and storage, suggesting that

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astaxanthin had no inhibitory effect on lactic acid bacteria.

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4. Conclusion

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In this study, the properties of microencapsulated astaxanthin and astaxanthin yogurt have

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been investigated. CMC-Na and MCC were able to encapsulate astaxanthin and increase the

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astaxanthin solubility, stability and antioxidation activity, as shown by the astaxanthin retention

315

rate and DPPH-scavenging activity in different conditions. After encapsulation, a slower change

316

was observed in the astaxanthin retention rate and the DPPH-scavenging activity of

15

ACCEPTED MANUSCRIPT microencapsulated astaxanthin. The microencapsulated astaxanthin was effectively dispersed in

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yogurt, leading to a slightly higher acidity than that of the plain one during fermentation and

319

storage. Overall results indicated that the microencapsulated astaxanthin could enhance the

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stability and antioxidation activity of yogurt as a food thickener, stabilizer and coloring additive

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and it might also have the potential use in other acid beverages.

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Acknowledgments

This research was supported by the State Key Laboratory of Pulp and Paper Engineering

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[2017TS06], the National Natural Science Foundation of China [grant nos. 51478190 and

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51278200] and Guangzhou Science and Technology Program [grant No.2014 Y2 -00515].

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astaxanthin on egg quality and growth of long snout seahorse (Hippocampus guttulatus)

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Yang, L., & Zhao, M. (1993). Research on improving the stability of yogurt. Journal of South

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China University of Technology, 4, 110-117. Yuan, C., Du, L., Jin, Z., & Xu, X. (2013). Storage stability and antioxidant activity of complex of astaxanthin with hydroxypropyl-beta-cyclodextrin. Carbohydr Polym, 91(1), 385-389. Zulkifli, N. I., Samat, N., Anuar, H., & Zainuddin, N. (2015). Mechanical properties and failure

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Design, 69, 114-123.

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ACCEPTED MANUSCRIPT Figures:

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Fig.1. Optical microscopy images（400×）of CMC-Na (A), MCC (B) and microencapsulated

444

astaxanthin (C); Microstructure by SEM (D. 500×, E. 2000×, F. 20000×) of microencapsulated

445

astaxanthin.

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Fig.2. FT-IR spectra of microencapsulated astaxanthin. (A) Microencapsulated astaxanthin,(B)

447

CMC-Na, (C) MCC, and (D) non-encapsulated astaxanthin.

448

Fig.3. Storage stability of microencapsulated astaxanthin (square) and non-encapsulated

449

astaxanthin (circle) at 55 ℃.

450

Fig.4. The pH stability 1.5 (A) and pH 6.8 (B) and DPPH-scavenging activity (C) of

451

microencapsulated astaxanthin (square) and non-encapsulated astaxanthin (circle).

452

Fig.5. Time course of yogurt (plain yogurt A: square ; astaxanthin yogurt A: circle; plain yogurt B:

453

upper triangle; astaxanthin B: lower triangle) fermentation and storage: DPPH-scavenging activity

454

(A, B), the total number of lactic acid bacteria (C, D), and acidity (E, F).

457 458 459 460

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461 462 463

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ACCEPTED MANUSCRIPT 464 465 466

A

B

C

D

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F

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Fig.1. Optical microscopy images（400×）of CMC-Na (A), MCC (B) and microencapsulated

471

astaxanthin (C); Microstructure by SEM (D. 500×, E. 2000×, F. 20000×) of microencapsulated

472

astaxanthin.

475 476 477 478

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ACCEPTED MANUSCRIPT 483

A

484 485

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B

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490 491 492 C 493

495 496

498 499 500 501

D

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502

Fig.2. FT-IR spectra of microencapsulated astaxanthin. (A) microencapsulated astaxanthin, (B)

503

CMC-Na, (C) MCC, and (D) non-encapsulated astaxanthin.

504

24

ACCEPTED MANUSCRIPT 505

100

80

60

40

0

4

8

12

Storage time (h)

20

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16

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Astaxanthin retention (%)

120

507

Fig.3. Storage stability of microencapsulated astaxanthin (square) and non-encapsulated

509

astaxanthin (circle) at 55 ℃. Results are expressed as mean value ± standard deviation of three

510

replicates.

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514 515 516 517 518

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519 520 521 522 25

ACCEPTED MANUSCRIPT 523 524 525

140

B

120

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A

130 100

110 100 90 80 70 60

80

60

40

50 0

1

2

3 4 Time (h)

5

110 100 90 80 70 60 50

1

2

3

4

5

6

40

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DPPH-scavenging activity (%)

0

Time (h)

526 C

20

6

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Astaxanthin retention (%)

Astaxanthin retention (%)

120

30 20 10

0

2

4

6

Storage time (h)

527

Fig.4. The pH stability 1.5 (A) and pH 6.8 (B) and DPPH-scavenging activity (C) of

529

microencapsulated astaxanthin (square) and non-encapsulated astaxanthin (circle). Results are

530

expressed as mean value ± standard deviation of three replicates.

532

AC C

531

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528

533 534 535

26

ACCEPTED MANUSCRIPT B

20 18 16 14 12 10

0

2

4

20

15

10

6

0

Fermentation time (h)

536

0

0

2

4

90 80

50

AC C

40

0

80 70 60 50 40 30 20 10

0

0

3

6

9

F 120 110 100 90 80 70 60

30 20

12

Storage time (d)

EP

70 60

6

TE D

100

9

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The total number of lactic acid bacteria (10^7 CFU/mL)

20

90

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Acidity (°T)

The total number of lactic acid bacteria (10^7 CFU/mL)

60

537

Acidity (°T)

6

D 80

Fermentation time (h)

E

3

Storge time (d)

C

2

4

50

6

Fermentation time (h)

538

25

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22

DPPH-scavenging activity (%)

DPPH-scavenging activity (%)

A 24

0

3

6

9

Storage time (d)

539

Fig.5. Time course of yogurt (plain yogurt A: square; astaxanthin yogurt A: circle; plain yogurt B:

540

upper triangle; astaxanthin B: lower triangle) fermentation and storage: DPPH-scavenging activity

541

(A, B), the total number of lactic acid bacteria (C, D), and acidity (E, F). Results are expressed as

542

mean value ± standard deviation of three replicates.

27

ACCEPTED MANUSCRIPT Tables:

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Table 1 Effect of different ratios of MCC to CMC-Na on the stability of inclusion complex

545

suspension

546

Table 2 Effect of pH on the stability of inclusion complex suspension (MCC: CMC-Na = 4:6)

547

Table 3 The properties (water solubility (%), encapsulated efficiency (EE, %), color parameters)

548

of microencapsulated astaxanthin.

549

Table 4 The stability and color of yogurt treated with the microencapsulated astaxanthin.

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550 551 552 553

557 558 559 560 561

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AC C

555

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562 563 564

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ACCEPTED MANUSCRIPT 565

Table 1 Effect of different ratios of MCC to CMC-Na on the stability of inclusion complex

566

suspension

567

pH 5:5

6.8

100% a

100% a

3.1

100% a

100% a

570 571

577 578

EP

576

AC C

575

TE D

572

574

7:3

100% a

61.85% b

60.15% b

53% b

53% b

59% b

Different letters indicate statistical differences between inclusion complex suspension (p < 0.05).

569

573

6:4

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4:6

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568

3:7

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MCC:CMC-Na

579 580 581 582

29

ACCEPTED MANUSCRIPT 583

Table 2 Effect of pH on the stability of inclusion complex suspension (MCC: CMC-Na = 4:6)

pH Time(d) 4.5

6.8

8.3

10.8

1

100% a

100% a

100% a

100% a

100% a

100% a

3

100% a

100% a

100% a

100% a

100% a

100% a

5

99% a

100% a

100% a

100% a

100% a

100% a

Different letters indicate statistical differences between inclusion complex suspension (p < 0.05).

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585 586 587 588

593 594 595

EP

592

AC C

591

TE D

589 590

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3.5

SC

584

2.8

596 597 598 599

30

ACCEPTED MANUSCRIPT 600

Table 3 The properties (water solubility (%), encapsulated efficiency (EE, %), color parameters)

601

of the microencapsulated astaxanthin.

602 Microencapsulated astaxanthin

Water solubility (%)

52.88±0.27

EE（%）

58.76±4.74

△L*

-5.4±0.56

△a*

5.44±5.40

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Properties

11.61±5.86

603

△L*: black (-) to white (+), △a*: green (-) to red (+), and △b*: blue (-) to yellow (+). Results are

604

expressed as mean value ± standard deviation of three replicates.

608 609 610 611

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607

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606

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605

612 613 614 615

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ACCEPTED MANUSCRIPT Table 4 The stability and color of yogurt treated with microencapsulated astaxanthin.

Properties

Control

Astaxanthin yogurt

Stability (%)

87.06±2.42a

96.47±0.45 b

L*

78.69±0.30 a

89.15±0.29 b

a*

-2.6±0.02 a

5.44±0.08 b

b*

4.92±0.07 a

11.61±0.15 b

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616

Control: without added astaxanthin; Astaxanthin yogurt: with the addition of 2 % micro-

618 619 620

encapsulated astaxanthin. L*: black (0) to white (100), a*: green (-) to red (+), and b*: blue (-) to yellow (+). Results are expressed as mean value ± standard deviation of three replicates. Different letters (a, b) indicate statistical differences between different yogurts (p < 0.05).

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621

AC C

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Highlights

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> A process for microencapsulation of astaxanthin by CMC-Na and MCC was developed >The stability of microencapsulated astaxanthin was significantly improved. >A new functional yogurt with encapsulated astaxanthin was developed.