Stirred-type yoghurt incorporated with sour cherry extract in chitosan-coated liposomes

Stirred-type yoghurt incorporated with sour cherry extract in chitosan-coated liposomes

Journal Pre-proof Stirred-type yoghurt incorporated with sour cherry extract in chitosan-coated liposomes Dila Akgün, Mine Gültekin-Özgüven, Aysun Yüc...

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Journal Pre-proof Stirred-type yoghurt incorporated with sour cherry extract in chitosan-coated liposomes Dila Akgün, Mine Gültekin-Özgüven, Aysun Yücetepe, Gokce Altin, Monika Gibis, Jochen Weiss, Beraat Özçelik PII:

S0268-005X(19)30941-5

DOI:

https://doi.org/10.1016/j.foodhyd.2019.105532

Reference:

FOOHYD 105532

To appear in:

Food Hydrocolloids

Received Date: 4 May 2019 Revised Date:

13 November 2019

Accepted Date: 18 November 2019

Please cite this article as: Akgün, D., Gültekin-Özgüven, M., Yücetepe, A., Altin, G., Gibis, M., Weiss, J., Özçelik, B., Stirred-type yoghurt incorporated with sour cherry extract in chitosan-coated liposomes, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.105532. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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Stirred-type Yoghurt Incorporated with Sour Cherry Extract in Chitosan-coated

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Liposomes

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Dila Akgüna, Mine Gültekin-Özgüvenb, Aysun Yücetepec, Gokce Altinb,d, Monika Gibisa,

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Jochen Weissa, Beraat Özçelikb,e *

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a

Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology,

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University of Hohenheim, Garbenstr. 21/25, 70599, Stuttgart, Germany b

Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering,

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Istanbul Technical University, Maslak, TR-34469, Istanbul, Turkey c

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Department of Food Engineering, Faculty of Engineering, Aksaray University, TR-

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68100, Aksaray, Turkey d

Molecular Engineering & Science Institute, University of Washington, 3946 W Stevens

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Way NE, 98105, Seattle, WA, US e

BIOACTIVE Research & Innovation Food Manufac. Indust. Trade Ltd., Katar Street, Teknokent ARI-3, B110, Sarıyer, TR-34467, Istanbul, Turkey

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*Corresponding Author: Prof. Dr. Beraat Özçelik

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Istanbul Technical University, Faculty of Chemical and Metallurgical Engineering,

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Department of Food Engineering, 34469, Maslak, Istanbul-TURKEY

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Telephone of office: +90 212 285 3933

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Fax number: +90 212 285 7333

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E-mail: [email protected]

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Running title: Encapsulated sour cherry extract in yoghurt 1

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Abstract

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Liposomal systems are promising carrier systems for the delivery of phenolic compounds.

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However, the commercial usage of aqueous liposomal dispersions is still challenging. In this

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study, an aqueous liposomal dispersion which was loaded with sour cherry phenolics (SCE)

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were converted into powder form by spray drying to make the use of such delivery systems

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industrially applicable. Then, the obtained SCE loaded liposomal powder was incorporated

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into a stirred-type yoghurt system. During 21-day storage at 4°C, sensorial and

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physicochemical properties such as pH, color, and whey syneresis of yoghurt with SCE

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loaded liposomal powder were investigated. According to the results, SCE was successfully

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encapsulated in aqueous liposomal dispersion and spray dried. Spray drying process did not

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degrade phenolic compounds that were encapsulated by liposomes. The structure of

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liposomes in the sample contained spray dried liposome encapsulates with SCE (Y-

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encapsulated SCE) was the most stable in yoghurt during storage which also affects the

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stability of SCE. Thus, SCE showed the lowest degradation level in this sample among all

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samples in terms of total phenolics and antioxidant capacity.This means that the SCE in

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yoghurt with liposomal encapsulates was protected during the storage period. The spray

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drying process also led to an increased total dry solid and reduced syneresis. Although

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syneresis of yoghurt containing liposomal powder with SCE and without SCE was the lowest,

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control yoghurt was chosen to be the best according to the sensorial evaluation.

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Key Words: Sour cherry polyphenols; Chitosan-coated liposomes; Stirred-type yoghurt;

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Spray drying; Storage stability

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2

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1. Introduction

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Liposomes are bilayer vesicles in which an aqueous volume is entirely surrounded by a

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phospholipid membrane. Thus, they are attractive encapsulation systems for water-soluble

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phenolic compounds (Rashidinejad, Birch, Sun-Waterhouse, & Everett, 2014).

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entrapment of phenolics in liposomes increase phenolics stability and maintain their

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biological activity against negative environmental conditions. Furthermore, liposomes provide

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pH and ionic strength stability to phenolics (Xu, Tanaka, & Czernuszka, 2007). Another

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advantage of liposomal encapsulation is to provide high loading capacity for partially or

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completely water-soluble components. Thus, the liposomal systems offer a better delivery

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platform for either hydrophilic or hydrophobic phenolic compounds. However, the aqueous

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liposomal dispersions have low kinetic and thermal stability (Altin, Gültekin-Özgüven, &

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Özçelik, 2018a). For this reason, converting the liposome dispersion into powder form is a

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more applicable delivery system for the industry (Karadag et al., 2013; Rashidinejad, Birch,

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Sun-Waterhouse, & Everett, 2014). In our previous study (Altin, Gültekin-Özgüven, &

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Özçelik, 2018b), it was showed that application of such delivery systems in spray dried form

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exhibited better results than dispersion form in ayran (drinking yoghurt). When the liposomal

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encapsulated cocoa phenolics incorporated into the ayran formulation via spray dried forms,

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the in-vitro bioaccessibility of cocoa phenolics increased approximately 2,5 folds compared

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with the cocoa phenolics incorporated by aqueous liposomal dispersion in terms of total

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phenolics and total flavonoids, and total antioxidant capacity.

The

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This study aimed to employ sour cherry (Prunus cerasus L.) fruit as the phenolic source.

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Sour cherries are rich in phenolic compounds, particularly anthocyanins which have been

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proved to possess several health-promoting benefits due to their antioxidant, anti-

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inflammatory, and anti-carcinogenic activities (Manach, Williamson, Morand, Scalbert, &

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Rémésy 2005; Pezzuto, 2008). A yoghurt system with low pH was chosen as a matrix for the

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addition of liposomes with maximum retention of plant-derived phenolics. Furthermore, 3

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yoghurt does not contain anthocyanins, hence it easier to follow the stability of added

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phenolics. In addition, yoghurt is one of the most popular and widely consumed products in

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health-food area all over the world. Therefore, there are a number of studies about yoghurt

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fortification using different plant extracts such as grape and callus extract (Karaaslan, Ozden,

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Vardin, & Turkoglu, 2011), tea extract (Najgebauer-Lejko, Sady, Grega, & Walczycka,

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2011), cinnamon extract (Helal, & Tagliazucchi) and extracts of different edible flowers

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(Pires, Dias, Barros, Barreira, Santos-Buelga, & Ferreira; 2018).

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By liposome encapsulation technique, interactions between phenolic compounds and milk

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proteins are reduced as well as the astringency of the added phenolic compounds is blocked

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(Altin, Gültekin-Özgüven, & Özçelik, 2018b). El-Said, El-Messery, & El-Din (2018)

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developed a fortified yoghurt containing encapsulated phenolic extract of doum extract

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powder in liposomes. They found that adding 5% of doum extract powder in liposome to the

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yoghurt formulation results in a product with characteristics similar to the control yoghurt but

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with higher antioxidant activity. Similarly, Ghorbanzade et al. (2017) fortified yoghurt

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formulation with nano-encapsulated fish oil, and they reported that nano-liposome

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encapsulation caused to increment of the total content as well as the stability of fish oil

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ingredients such as dokosaheksaenoik asit (DHA) and eicosapentaenoic acid (EPA). In this

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concept, freeze dried SCE, liposomal powder with SCE, liposomal powder without SCE and

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SCE powder were incorporated into yoghurt with the a ratio of 5% (w/v) stored at 4°C for a

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shelf-life of 21 days. Samples were collected at 7 day-intervals (1st, 7th, and 14th and 21st day).

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Particle size distribution and ζ-potential were determined for characterization of primary

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(uncoated) and secondary (coated) liposomes, and liposomal powder. Scanning Electron

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Microscope (SEM) images were also taken for liposomal powder. The aims of this study were

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i) to investigate physico-chemical stability of liposomal powder with sour cherry extract by

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total phenolic content and antioxidant capacity (CUPRAC assay), ii) to understand how

4

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liposomal systems are applicable to a food system (stirred-type yoghurt) in terms of sensorial

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properties, color, pH, and whey syneresis. To our knowledge, this is the first report on the

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addition of spray dried liposomal powder containing SCE into a yoghurt system and

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observation of the changes of sensorial properties and characteristics of the new product.

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2. Materials and methods

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2.1 Materials

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Sour cherry concentrate with 65 °Brix was kindly provided by the Turkish Fruit Juice

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Industry Association (MEYED) (Istanbul, Turkey). Freeze-dried commercial yoghurt culture

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(YC-350, 50-U pouches) was a gift from Chr. Hansen (Istanbul, Turkey). Skim milk powder

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was manufactured by Pinar Süt Mamulleri San. A.Ş. (Izmir, Turkey). Lecithin (Soybean

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phospholipids, 97%- Ultralec® P) was obtained from Rotel (Istanbul, Turkey). Chitosan (TM

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3790, 79% degree of deacetylation) was granted by Primex® (Siglufjordur, Iceland). Acetone

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(purity ≥ 99.5%), hydrochloric acid, potassium chloride, sodium hydroxide, and gallic acid

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were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Triton X100,

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glacial acetic acid, sodium acetate trihydrate, and Folin-Ciocalteu reagent were purchased

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from Merck KGaA (Merck, Darmstadt, Germany). 20 DE value of Maltodextrin (MD) was

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provided from Tunckaya Kimyevi Maddeler Ticaret ve Sanayi Inc., Turkey. Sephadex G-50

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was supplied from GE Healthcare Life Sciences (Uppsala, Sweeden).

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2.2 Extraction of polyphenols from sour cherry concentrate

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In brief, 10 mL of sour cherry concentrate was mixed with 100 mL of 70% methanol and

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stirred for 15 min for 3 times. Methanol was removed under reduced pressure using a rotary

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evaporator (Bibby Sterilin RE-100, Bibby Scientific Limited, Staffordshire, UK) at 40 °C

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until the acetone had been removed. The remaining aqueous extract was collected and freeze

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dried (Alpha 1-2 LDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz,

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Germany).

5

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2.3 Preparation of primary liposomes and secondary liposomes

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A lecithin dispersion (2%, w/w) in acetate buffer (pH 3.5 ± 0.1; 0.1 M) was prepared. Then,

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freeze dried sour cherry extract (SCE) (0.2%, w/w) was dissolved in lecithin solution. For

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homogenization of the lecithin dispersion, a high shear disperser (DI-25 Yellowline, IKA)

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was employed for 10 min at 9500 rpm. A high pressure homogenizer (Microfluidizer

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Processor M-110L, Microfluidics, Newton, USA) was operated at homogenization pressure of

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25,000 psi to obtain primary liposomes without SCE and with SCE (0.2%, w/w). Lecithin

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solutions with and without SCE were passed five times through a high pressure homogenizer.

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The chamber of homogenizer was cooled during the homogenization with ice water to prevent

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the heating of samples. Secondary liposomes were produced via layer-by-layer deposition

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method. For this aim, liposomes with and without SCE were added to chitosan solutions

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(0.4%, w/v) in acetate buffer solution (pH = 3.5 ± 0.1; 0.1 M) stirred overnight at room

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temperature. Therefore, the surface of negatively charged primary liposomes was covered

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with a positively charged chitosan layer.

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2.4 Spray drying of secondary liposomes

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MD was used as a drying aid to facilitate spray drying. The mixtures of 20 % (w/w) MD, 0.5

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% (w/w) lecithin, 0.2 % (w/w) chitosan, and 0.05% (w/w) SCE, secondary liposomal

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dispersions with and without SCE were prepared. To use SCE powder (0.05%, w/w) as a

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blank, SCE (0.1%, w/w) was dissolved in acetate buffer, mixed with MD (1:1) (40% w/v in

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acetate buffer). Similarly, secondary liposomal dispersions were mixed with MD (1:1) and

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stirred overnight at room temperature. Prepared liposomal dispersions and SCE solution in

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MD were spray dried using a laboratory scale spray dryer (Mini Spray Dryer B-290, Büchi,

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Switzerland). The spray dryer was operated with a 1.5-mm nozzle atomizer at an atomizing

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air flow of 5 cm3/min. The drying condition was 2.5 cm3/min of feed rate, 160 °C at inlet

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temperature, 90 °C at outlet temperature and 0.67 m3/min air flow. Collected dried powders

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were placed in a desiccator at room temperature and stored in airtight containers. A UniBloc

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Moisture Analyzer MOC63u was used for the determination of the residual water content of

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powders.

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2.5 Characterization of primary and secondary liposomes, and liposomal powder

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The liposomes in the dispersions were characterized by measuring the ζ -potential and z-

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average mean diameter immediately after production. The ζ-potential was determined using a

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particle charge titration analyzer (Stabino®, Microtrac Europe, Montgomeryville, PA, USA).

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The particle size distribution was determined by a static light scattering instrument

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(Mastersizer MS2000, Malvern Instruments, Worcestershire, UK). The average particle

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diameters were calculated via the volume mean diameter (

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powder, 0.5 g powder was dissolved in 4.5 mL of acetate buffer. After stirring overnight, the

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particle size and ζ-potential were analyzed using the above mentioned method.

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2.6 SEM analysis of liposomal powder

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SEM analysis was carried out using a FEG 250 scanning electron microscope (FEI, USA).

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Prior to analysis, the samples were gold sputter-coated in a Leica vacuum coating unit in

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order to prevent the charging of the specimen. Operating conditions such as accelerating

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voltage, magnification, and working distance are indicated on the SEM images.

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2.7 Yoghurt manufacturing and incorporation of liposomal powder with SCE

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For stirred‐type yoghurt manufacturing, yoghurts were formulated using a skim milk powder

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with 1.25% (w/w) fat content which diluted in distilled water to a total solid content of 13%

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(w/v). Then, they were placed in heat treated glass jars at 95 °C for 15 min in the water bath.

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After cooling to the fermentation temperature (42 °C), the mixtures were subsequently

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incubated with the bacterial culture (0.4%, v/v) containing Lactobacillus delbrueckii subsp.

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bulgaricus and Streptococcus thermophilus and incubated in the water bath at 42 °C until pH

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4.60 was reached (Erşan et al., 2016). The freeze dried SCE, liposomal powder with SCE and 7

4,3).

To reconstite the spray-dried

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SCE powder were added to yoghurt formulations with a ratio of 5% (w/w). To eliminate the

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inferents during experiments, control samples (plain yoghurt and yoghurt containing

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liposomal powder without SCE) were also prepared. Then, yoghurt samples were stored at 4

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°C for 21 days. Samples were collected on the 1sh, 7th, 14th, and 21st days of storage. Yoghurt

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sample codes were as follows: Control yoghurt, yoghurt sample containing freeze dried SCE

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(Y-freeze dried SCE), yoghurt sample containing spray dried SCE (Y-spray dried SCE),

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yoghurt sample containing blank liposomal powder (Y-blank encapsulate), yoghurt sample

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containing liposomal powder with SCE (Y-encapsulated SCE).

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2.8 pH

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The pH of the samples was determined using a digital pH meter (Hanna pH 211 Model pH

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meter, USA). All measurements were performed in triplicates.

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2.9 Color

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The color parameters L*, a*, b* values were measured by using a colorimeter (CR 400,

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Minolta, Japan). A white tile was used for standardization. All measurements were performed

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in triplicates. Whiteness Index (WI), hue and chroma parameters of the samples were

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calculated using the equations below (Vargas et al., 2008):

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WI = 100 - [(100 - L*)2+ a*2+ b*2]1/2

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Hue = h*ab = tan ( ∗ )

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Chroma = C∗ = (a ∗ + b ∗ )

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2.10 Whey syneresis (%) measurement

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The amount of syneresis was determined by calculating water holding capacity (WHC),

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according to Keogh and O’Kennedy (1998). Briefly, 20 g of yogurt was centrifuged for 10

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min at 4000 rpm at 20 °C. The supernatant was removed and the centrifuge tube containing

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sediment was weighed. All analyses were performed in triplicates. The water absorption

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capacity was determined by the below equation:

(1)



(2) .

(3)

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(

)=

× 100

is the weight of yogurt (g),

(4)

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where;

the weight of the tube plus yogurt (g) and

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weight of the tube plus the sediment (g).

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2.11 Preparation of yoghurt extracts

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Freshyl collected yoghurt samples were extracted in defined days (1st, 7th, 14th, and 21st day)

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to determine the stability of encapsulated SCE in yoghurt formulation according to the

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method of de Carvalho et al. (2018). Briefly, 20 g of yoghurt sample was treated with 30 ml

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of acidified acetone solution, was vortexed, and kept at + 4°C overnight. After filtrating

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through Whatman No:2 filter paper, acetone was evaporated from the collected aqueous phase

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at 40 oC by using rotary evaporator. Following centrifugation at 10,000 rpm for 2 min,

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collected top-aqueous phase was freeze dried and kept at -80 °C.

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2.12 Determination of total phenolic content

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The total phenolic content (TPC) was measured by the Folin-Ciocalteu assay according to the

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method of Gibis, Vogt & Weiss (2012). Diluted sample (1 ml) was treated with Folin–

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Ciocalteau reagent (5 ml) for 3 min. After the addition of sodium carbonate solution (4 ml),

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the mixture was left to stand in dark for 60 min. The absorbance of all samples was measured

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at 720 nm. Triplicate analyses were performed for each analysis. Results were expressed as

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mg gallic acid equivalents (GAE) per L sample. Sephadex gel filtration was applied to remove

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chitosan which did not attach to the liposome surface and the free extract which was not

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encapsulated in liposomes as described by Gültekin-Özgüven et al. (2016), previously.

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2.13 Determination of total antioxidant capacity

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Total antioxidant capacity (TAC) was performed according to the Cupric ion reducing

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antioxidant capacity (CUPRAC) method described by Apak et al. (2014). In brief, 100 µl of

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diluted sample was mixed with 1 ml of ammonium acetate (pH:7), 1 ml of neocuproine

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solution (7,5x10-3M), 1 ml of copper (II) chloride solution (10-2 mM) and 1 ml of MQ water, 9

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respectively. After keeping in dark for 25 min, the mixture was centrifuged at 300 rpm for 5

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minutes. The extinction was measured at 450 nm. The results were expressed as mg trolox per

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100 g sample.

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2.14 Sensory analysis

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The Quantitative Descriptive Profile (QDP) Method was used to conduct sensory analysis (BS

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EN ISO 13299, 2016). For sensory analysis, selected 10 members, master and Ph.D. students

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(ages of 22- 50 years old) of the Food Engineering Department were trained to carry out the

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sensory analyses at 1st, 7th, 14th and 21st storage days. The selected assessors worked under the

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direction of the panel leader to develop a sensory language and describe the sensory

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properties of the products. Samples were evaluated by these trained panelists in terms of

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color, dairy sour, fruity, general appearance, gel firmness, lumpiness, off-odour, and whey

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separation. The degrees of sensory quality scores were between 1 and 5 for each category,

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where 1: unacceptable, 2: hardly acceptable, 3: acceptable, 4: good and 5: perfect.

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2.15 Statistical analysis

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The general linear model procedure was used to investigate the treatment and interaction

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effects via a statistical program (Minitab, Version 17, Minitab Inc., State College, PA). Tukey

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test was used to determine the differences between mean values. p value of <0.05 was chosen

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to determine significant differences. All analyses were repeated at least three times using

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triplicate samples.

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3. Results and Discussion

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3.1 Characterization of primary and secondary liposomes, and liposomal powder

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The mean particle diameter of primary liposomes without SCE was found approximately 146

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nm after the homogenization at 22.500 psi. The ζ-potential of initially anionic primary

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liposomes was determined -25.4 mV. Various chitosan concentrations (0.1 – 0.5%, w/w) were

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added the anionic primary liposome dispersion to find the optimal chitosan concentration to

10

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coat the primary liposomes. The effect of chitosan concentration on ζ-potential and mean

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particle diameter was observed. When the surface charge had reached an almost constant

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value (~40 mV), the lowest mean diameter (~303 nm) was observed at a chitosan

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concentration of 0.4% (w/w). Based on these results, we continued our studies with an

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optimal chitosan concentration of 0.4% (w/w) (Table 1). As we reported before (Gültekin-

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Özgüven et al., 2016) addition of MD to primary liposomes caused a complete breakdown of

254

the system due to extensive flocculation, and these systems were thus unsuitable for the

255

subsequent spray-drying process. Hence, only secondary blank liposomes and secondary

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liposomes with SCE (0.1%, w/w) were mixed with MD (40%, w/w) and then spray dried. As

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seen in Table 1, the particle size of reconstituted samples were similar or slightly smaller than

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before spray drying (p > 0.05) owing to the potential reducing water activity of salts and

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sugars. The concentration gradient between the inside and outside of the liposomes might

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change after the addition of MD. The water in the core of liposome may migrate after mixing

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with the MD, hence the concentration gradient was decreased. Thus, the reduction of particle

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size may be explained by the osmotic driving force (Karadag et al., 2013). In addition,

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moisture content of samples was 73.89 ± 2.06, 75.92 ± 2.35, and 76.71 ± 1.89 for liposomal

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powder without and with SCE, and SCE powder, respectively. There was no significant

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difference between the detected samples (p > 0.05) in terms of moisture content.

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3.2 Content and location of SCE in primary and secondary liposomes, and liposomal

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powder

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Location of phenolics can be the interior of liposomes, partially in the phospholipid

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membrane or onto the surface of liposomes. The primary liposomes containing 0.1% (w/w)

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extract showed the highest encapsulation efficiency (71.2%), which means that 71.2% of the

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SCE was attached to the surface and the interior of the primary liposomes. Our results are

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comparable with the results reported before by Gibis, Vogt & Weiss (2012) (83.5% for grape

11

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seed extract 0.1%, w/w). In this study, 12.81 mg/L of phenolic substances were detected on

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the surface of the liposomes after gel filtration. In order to calculate the phenolic content

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inside the liposomes, the concentration detected after gel filtration was subtracted from the

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concentration detected after Triton treatment of filtered samples. This yielded concentration

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of 14.41 mg/L. The efficiency of phenolic substances incorporation in secondary liposomes

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with SCE was found at 78.5%. In this case, the phenolic content on the surface was detected

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only 9.5 mg/L and the content in the interior of the liposomes was determined that 15.31

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mg/L. This indicates that coating the liposome surface with chitosan polymer makes the

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phenolics less accessible. While TPC was found to be 45.82 mg/L for the pure extract (0.1%,

282

w/w) dissolved in acetate buffer, TPC of primary liposomes containing 0.1% (w/w) SCE was

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found that 38.19 mg/L. However, this value decreased to 31.23 mg/L after coating with

284

chitosan. This loss can be explained by the fact that existing of chitosan might block the

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accessibility of phenolics inside or on the surface of primary liposomes (Gibis, Vogt & Weiss,

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2012). After spray drying of secondary liposomes with SCE, the total amount of detectable

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phenolics was found that 37.52 ± 1.13 mg/L. Compared to the results of TPC of liposomal

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dispersions (38.19 mg/L) before spray drying, it was observed that spray drying process did

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not lead to significant decreases in phenolics. In contrast, SCE powder showed a significant

290

decrease in TPC (29.85 ± 1.56 mg/L). This can be explained that spray drying process did not

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degrade phenolic compounds encapsulated in liposomes due to the protective function of both

292

the liposomal membrane and the biopolymer coat.

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3.3 Powder morphology of liposomal powders

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Liposomal powders were imaged by Scanning Electron Microscope (SEM). According to

295

Figure 1, whether liposomal powders without SCE (A series) contained some small

296

indentations and wrinkles on their surfaces, they were mostly spherical. Their diameter ranged

297

was detected between 1 and 5 µm. Similarly, liposomal powders with SCE (B series)

12

298

contained particles with a similar range of diameter, and encapsulation of SCE did not affect

299

the overall surface morphology. In general, larger particles appeared more collapsed and

300

broken with deeper indentations. Such surface morphology has been also reported in other

301

studies (Karadag et al., 2013; Lo, Tsai, & Kuo, 2004; Tonon, Freitas, Hubinger, 2011).

302

According to Nijdam and Langrish (2006), dent formation is related to a formed vacuole

303

within the particle soon after skin development on the surface. As the particle temperature

304

exceeds the local ambient boiling point, the vacuole inflates and the vapor pressure within the

305

vacuole rises above the local ambient pressure. The moisture evaporates very quickly when

306

the drying temperature is sufficiently high, and the skin becomes dry and hard. For this

307

reason, as the particle moves to the cooler regions of the drier, the vapor can not retain its

308

hollow particulate when concentrated in the vacuum. However, when the drying temperature

309

is lower, the skin is being moist and stretched for a longer time, so that the hollow particle can

310

deflate and shrivel as it cools.

311

3.4 pH of yoghurt samples

312

Change in pH of yogurt samples was evaluated during 21 days of storage (Figure 2A). The pH

313

value of all samples decreased from approximately 4.5 to 4.0 at the end of the storage due to

314

the microbial activity of lactic acid bacteria. The decrease in pH was found to be significantly

315

different for all samples collected on the 14th and 21st days. In comparison to Ghorbanzade et

316

al. (2017) who reported a lower pH in control yoghurt than fortified yoghurt with nano-

317

encapsulated fish oil (in dispersion form), no significant difference was observed among

318

samples. This adverse position may be explained that the addition of encapsulated SCE in

319

powder form.

320

3.5 Syneresis of yoghurt samples

321

Change in syneresis of yoghurt samples during 21 days of storage is shown in Figure 2B. It

322

was observed that the amount of water released in each sample did not change up to 14 days.

13

323

It was observed that syneresis became reduced during the storage period. Especially, syneresis

324

was remarkably in a lower amount the last week of storage compared to first two weeks. Due

325

to contracting effect on the casein micelle matrix by pH reduction led to be more serum to be

326

released during storage. A similar observation was reported by Ghorbanzade et al. (2017).

327

Compared to control yoghurt and Y-freeze dried SCE, the amount of syneresis of the yoghurt

328

samples which contained spray dried ingredient (spray dried SCE, blank spray dried

329

liposomal powder and spray dried liposomal powder with SCE) was found lower amount. On

330

the other hand, the syneresis of control yoghurt and Y-freeze dried SCE were detected in

331

similar value while Y-blank encapsulates, Y-encapsulated SCE, and Y-spray dried SCE had a

332

similar amount of syneresis. Since spray drying treatment may be resulting in an increased

333

total dry solid and water holding capacity can reduce syneresis. Although not reported here,

334

lecithin’s retarding effect on syneresis may be possible (Ghorbanzade et al., 2017).

335

3.6 Color evaluation of yoghurt samples

336

Change in the luminosity (L*), chroma (C*ab), hue (h*ab) and whiteness index (WI) of all

337

yoghurt samples during storage is given in Figure 3. Compared to control yoghurt, the

338

addition of liposomal powders and SCE did not change color parameters (L*, WI chroma, and

339

hue) significantly up to the 14th day of storage. However, these parameters decreased

340

dramatically for all yoghurt samples in the last week of storage time significantly.

341

3.7 Sensory evaluation of yoghurt samples

342

Mean scores of the sensory evaluation parameters were statistically analyzed and the results

343

are given in Table 2. On the first day, all samples were similar in terms of off-odour, color,

344

syneresis, gel firmness, dairy sour, and general appearance scores. Only, the lumpiness score

345

of control yoghurt and Y-freeze dried SCE, and the fruity score of Y-freeze dried SCE and Y-

346

blank encapsulate was different from each other. Except the Y-blank encapsulate, which

347

exhibited the lowest value, all samples had similar color scores. At the end of storage (21st 14

348

day) gel firmness, lumpiness, general appearance, fruity and dairy sour scores were similar

349

among all samples. In terms of off-odour, Y-blank encapsulate was different from control

350

yoghurt in desired manner. The color of Y-blank encapsulate was negatively different from

351

Y-encapsulated SCE. In contrast to syneresis measurement, control yoghurt was determined

352

to be the best compared to Y-freeze dried SCE, Y-spray dried SCE, Y-encapsulated SCE and

353

Y-blank encapsulate regarding sensory analysis, which was an unexpected situation. On the

354

other hand, the off-odour score of control yoghurt and Y-spray dried SCE, lumpiness score of

355

control yoghurt, fruity score of control yoghurt and Y-blank encapsulate, color score of Y-

356

spray dried SCE and Y-encapsulated SCE did not change during storage period. Furthermore,

357

the color score of control yoghurt started decreasing in the 14th day while that of Y-blank

358

encapsulate decreased at the 21st day during storage. Dairy sour and general appearance

359

scores of all samples maintained during storage.

360

3.8 Physico-chemical stability of SCE encapsulated with liposomal powder in yoghurt

361

Physico-chemical stability of SCE encapsulated by liposomal powder in yoghurt formulation

362

was observed via total phenolic content (TPC) and antioxidant capacity (TAC) in defined

363

days (1st, 7th, 14th, 21st day) (Figure 4). It was detected that control yoghurt, Y-freeze dried

364

SCE, and Y-spray dried SCE contained similar amounts of phenolics whereas Y-encapsulated

365

SCE had the highest phenolic content in the 1st day. It was detected no significant difference

366

(p > 0.05) between control yoghurt and Y-spray dried SCE on the 7th and 14th days, in terms

367

of TPC amount. Meanwhile, on day 7 Y-freeze dried SCE exhibited the highest TPC where

368

Y-encapsulated SCE was following it. In contrast, Y-encapsulated SCE had the highest TPC

369

followed by Y-freeze dried SCE on the 14th day. At the end of storage, Y-spray dried SCE

370

and Y-freeze dried SCE had a similar amount of TPC (p> 0.05). Y-encapsulated SCE had the

371

highest and control yoghurt had the lowest regarding TPC. On the other hand, TPC value of

372

Y-encapsulated SCE and Y-spray dried SCE were different among the storage periods. In 15

373

both sample sets, TPC decreased on the 1st and 7th days, it increased on the 14th day, and again

374

decreased on the 21st day. The TPC amount of control yoghurt did not change between the 1st

375

and 7th days, but increased on the 14th day, and decreased on the 21st day. For Y-freeze dried

376

SCE, TPC amount increased on the 7th day, did not change between the 7th and 14th days,

377

decreased on the 21st day. All in all, TPC value followed the order: Y-encapsulated SCE> Y-

378

spray dried SCE = control yoghurt = Y-freeze dried SCE in the 1st day, Y-freeze dried SCE >

379

Y-encapsulated SCE > control yoghurt = Y-spray dried SCE in the 7th day, Y-encapsulated

380

SCE > Y-freeze dried SCE > control yoghurt = Y-spray dried SCE in the 14th day, Y-

381

encapsulated SCE > Y-freeze dried SCE = Y-spray dried SCE > control yoghurt in the 21st

382

day. The interaction ability of phenolic compounds with milk proteins in dairy products

383

affects their specific functionality. Since the proteins have achieved the isoelectric points at

384

the pH of produced yoghurt (pH: 4.6), the protein−polyphenol interaction is observed as

385

maximal (Trigueros et al., 2014). For this reason, it was not possible to see a significant

386

difference between control yoghurt and Y-freeze dried SCE at the beginning. During storage,

387

this interaction decreased depending on pH reduction. Consequently, the TPC of Y-freeze

388

dried SCE was determined to be higher than control yoghurt. Showing a similar trend in TPC,

389

the antioxidant capacity of all samples determined by CUPRAC assay were similar up to the

390

7th day, increased in the 14th day, but decreased in the 21st day. This decrease was dramatical

391

for control yoghurt, Y-freeze dried SCE, and Y-spray dried SCE after a week. In contrast, the

392

antioxidant capacity of SCE in Y-encapsulated SCE was protected during storage.

393

Antioxidant capacity of samples followed the order: Y-encapsulated SCE > Y-freeze dried

394

SCE = Y-spray dried SCE > control yoghurt in the 1st day, Y-encapsulated SCE > Y-spray

395

dried SCE > Y-freeze dried SCE > control yoghurt in the 7th, 14th, and 21st days. The

396

interaction of phenolics with milk proteins did not affect the antioxidant capacity of yogurt as

397

reported by Trigueros et al. (2014) previously. 16

398

4. Conclusion

399

In conclusion, it is obvious that the liposome structure did not change and SCE was stable in

400

Y-encapsulated SCE sample among all yoghurt samples during storage. Therefore, it is

401

possible to say that liposomal encapsulation provided SCE stability in terms of TPC and

402

TAC. Moreover, the spray drying process did not damage the liposome structure as well as

403

did not degrade SCE phenolics. These findings showed that this system was a suitable

404

phenolic delivery method through yoghurt product. Although Y-spray dried SCE sample was

405

employed as a control sample in this study, spray drying also served as a microencapsulation

406

technique. As a result, SCE in Y-spray dried SCE sample was protected better than Y-freeze

407

dried SCE, but not as much as Y-encapsulated SCE. As expected, phenolic concentration of

408

Y-freeze dried SCE degraded faster during 21 days.

409

Acknowledgments

410

This project was financially supported by the Erasmus Scholarship for European Internship in

411

Research and Development.

412 413

17

414

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415

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Helal, A., & Tagliazucchi, D. (2018). Impact of in-vitro gastro-pancreatic digestion on

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Rashidinejad, A., Birch, E. J., Sun-Waterhouse, D., & Everett, D. W. (2014). Delivery of

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hard cheese. Food Chemistry, 156, 176-183.

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Tonon, R.V., Freitas, S. S. & Hubinger, M. D. (2011). Spray Drying of acai (Euterpe

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Trigueros, L., Wojdyło, A., & Sendra, E. (2014). Antioxidant activity and protein–polyphenol

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interactions in a pomegranate (Punica granatum L.) yogurt. Journal of Agricultural

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485

Vargas, M., Cháfer, M., Albors, A., Chiralt, A., & González-Martínez, C. (2008).

486

Physicochemical and sensory characteristics of yoghurt produced from mixtures of

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490

21

491

Figure captions

492

Figure 1. SEM images of liposomal powders (A: secondary liposomes without SCE; B:

493

secondary liposomes with SCE). Pictures were taken at 4.000X and 10.000X magnifications,

494

respectively.

495

Figure 2. Change in pH (A)* and syneresis (%) (B) of control yoghurt , yoghurt sample

496

containing freeze dried SCE (Y-freeze dried SCE) , yoghurt sample containing blank

497

liposomal powder (Y-blank encapsule), yoghurt sample containing liposomal powder with

498

SCE (Y-encapsulated SCE), yoghurt sample containing spray dried SCE (Y-spray dried SCE)

499

during 21 days of storage at 4°C.

500

Figure 3. Change in color parameters of control yoghurt , yoghurt sample containing freeze

501

dried SCE (Y-freeze dried SCE) , yoghurt sample containing blank liposomal powder (Y-

502

blank encapsule), yoghurt sample containing liposomal powder with SCE (Y-encapsulated

503

SCE) , yoghurt sample containing spray dried SCE (Y-spray dried SCE) during 21 days of

504

storage at 4°C.

505

Figure 4. Change in TPC and antioxidant capacity of control yoghurt , yoghurt sample

506

containing freeze dried SCE (Y-freeze dried SCE) , yoghurt sample containing blank

507

liposomal powder (Y-blank encapsule), yoghurt sample containing liposomal powder with

508

SCE (Y-encapsulated SCE) , yoghurt sample containing spray dried SCE (Y-spray dried

509

SCE) during 21 days of storage at 4°C.

510 511

22

Table 1. Mean particle diameter and ζ-potential of secondary liposomes without and with SCE (0.1%, w/w) and of SCE solution and after reconstitution of powders. Initial mean

Mean diameter of

Initial ζ-potential

ζ-potential after

diameter (nm)

reconstituted

(mV)

reconstitution of

liposomes (nm)

Secondary liposome

powders (mV)

303.25 ± 4.22 a, A

271.80 ± 5.40 b, A

40.38 ± 4.10 a, A

32.8 ± 3.05 a, A

342.00 ± 9.33 a, A

276.25 ± 4.65 b, A

43.66 ± 3.52 a, A

33.8 ± 2.36 a, A

-

-

-16.33 ± 1.56 a, B

- 11.91 ± 3.52 a, B

without SCE Secondary liposome with SCE SCE

*Values with different superscript small letters within the row are significantly different. Values with different superscript capital letters within the column are significantly different (p < 0.05).

Table 2. Change in sensorial properties of control yoghurt , yoghurt sample containing freeze dried SCE (Y-freeze dried SCE) (198), yoghurt sample containing blank liposomal powder (Y-blank encapsule) (642), yoghurt sample containing liposomal powder with SCE (Y-encapsulated SCE) (527), yoghurt sample containing spray dried SCE (Y-spray dried SCE) (379) during 21 days of storage at 4°C.

Sample

Storage day

Property 1

198 (Y-freeze dried SCE)

14 a,A

21 a,B

2.4±1.4a,B

0.6±0.5

0.8±0.8

2.0±2.6

Color

2.6±0.5a,A

1.8±0.4ab,A

1.0±0.0b,C

1.2±0.8b,AB

Whey separation

3.6±1.0ab,AB

4.8±0.5a,ABC

3.0±0.7b,C

4.8±0.5a,A

Gel firmness

4.4±0.8b,A

5.4±0.5a,A

2.8±0.4c,A

4.4±0.8ab,A

Lumpiness

4.0±0.0a,B

4.0±0.0a,C

3.4±0.5a,C

4.0±1.1a,A

Dairy sour

3.8±1.0a,A

4.4±1.2a,A

5.4±1.0a,A

5.0±1.1a,A

Fruity

0.8±0.8a,AB

0.6±0.5a,A

0.0±0.0a,A

0.0±0.0a,A

General apperance

4.8±1.2ab,A

5.8±0.4a,B

3.2±1.9b,A

4.0±0.6ab,A

Off-odour

0.6±0.5bc,A

0.4±0.5c,A

2.8±1.5ab,AB

3.6±1.5a,AB

Color

2.6±0.5a,A

2.0±0.0ab,A 2.0±0.6ab,ABC

1.6±0.5b,AB

Whey separation

5.0±0.7b,AB

6.6±0.6a,C

5.0±0.7b,AB

2.8±0.4c,CD

Gel firmness

4.0±1.2a,A

3.4±0.5a,AB

3.8±1.0a,A

4.2±1.2a,A

Lumpiness

5.6±0.7a,A

6.0±0.6a,A

5.0±0.6a,AB

2.8±1.2b,A

Off-odour

147 (Control yoghurt)

7 a,A

Dairy sour

4.6±0.7a,A

4.4±0.8a,A

5.2±0.8a,A

5.0±0.6a,A

Fruity

2.6±1.4a,A

0.4±0.5b,A

0.0±0.0b,A

0.4±0.8b,A

General apperance

4.4±0.7a,A

4.8±1.0a,AB

3.8±2.0a,A

4.2±0.4a,A

Off-odour

0.8±0.7b,A

2.4±1.7ab,A

3.2±2.0ab,AB

4.6±1.5a,AB

Color

2.4±0.5a,A

2.8±0.4a,A

3.2±1.0a,A

3.0±0.9a,A

Whey separation

3.6±1.1a,AB

3.8±0.5a,A

3.2±0.5a,C

2.4±0.9a,CD

527

Gel firmness

2.8±0.7a,A

4.4±1.2a,AB

4.4±1.2a,A

4.8±0.4a,A

(Y-encapsulated SCE)

Lumpiness

4.0±0.0b,B

5.8±0.4a,AB

5.2±0.4a,A

2.6±0.8c,A

Dairy sour

4.6±0.9a,A

5.2±1.0a,A

5.4±0.5a,A

5.6±1.0a,A

Fruity

1.0±0.8a,AB

0.2±0.4b,A

0.0±0.0b,A

0.2±0.4b,A

General apperance

3.6±0.7a,A

4.0±1.3a,A

3.0±1.1a,A

4.0±0.6a,A

Off-odour

1.0±1.0b,A

2.0±1.7b,A

3.0±2.3ab,AB

5.8±1.0a,A

Color

2.6±0.5a,A

2.8±0.4a,A

3.0±0.6a,A

1.4±0.5b,B

Whey separation

3.2±0.8b,B

4.6±0.5a,ABC

3.6±0.5ab,BC

3.0±0.6b,CD

642

Gel firmness

4.4±0.7a,A

5.0±1.1a,AB

4.2±1.3a,A

4.2±0.8a,A

(Y-blank encapsule)

Lumpiness

5.0±0.8a,AB

5.4±0.5a,AB

4.8±0.8a,AB

3.0±1.1b,A

Dairy sour

4.8±0.7a,A

4.6±0.5a,A

6.0±1.1a,A

5.8±0.4a,A

Fruity

0.4±0.5a,B

0.0±0.0a,A

0.0±0.0a,A

0.0±0.0a,A

General apperance

3.6±0.7a,A

4.2±1.0a,AB

3.4±2.1a,A

4.0±0.6a,A

Off-odour

0.8±0.8a,A

0.6±0.8a,A

2.8±2.1a,AB

3.6±1.3a,AB

Color

2.6±0.5a,A

2.8±0.4a,A

2.6±0.5a,A

2.2±1.0a,AB

Whey separation

4.6±1.0a,AB

4.2±0.8a,A

3.4±0.9a,C

3.6±1.3a,BCD

379

Gel firmness

4.2±1.2a,A

4.8±0.8a,AB

3.6±0.8a,A

4.0±0.6a,A

(Y-spray dried SCE)

Lumpiness

5.2±0.8a,AB

5.6±0.5a,AB

4.0±0.0ab,BC

3.4±1.4b,A

Dairy sour

4.2±1.0a,A

4.6±0.5a,A

5.4±1.0a,A

5.4±1.4a,A

Fruity

1.0±0.9a,AB

0.2±0.4b,A

0.0±0.0b,A

0.0±0.0b,A

3.6±0.8a,A 4.2±1.0a,AB 3.4±2.0a,A 4.2±1.2a,A General apperance *Values with different superscript small letters within the row are significantly different. Values with different superscript capital letters within the column are significantly different (p < 0.05).

A

B

Figure 1. SEM images of liposomal powders (A: Blank liposomal encapsulates; B: Liposomal encapsulates with SCE). Pictures were taken at 4.000X and 10.000X magnifications, respectively.

7th day

14th day

21st day

A

PH

1st day

Control Yoghurt

Y-freeze dried SCE

Y-blank encapsulate

Y-encapsulated SCE

Y-spray dried SCE

80,00

B

70,00

Syneresis (%)

60,00 50,00 40,00 30,00 20,00 10,00 0,00 1

7

14

21

Storage period (days), 1-7-14-21 Control Yoghurt 147

Y-freeze dried SCE

198

Y-blank 642

encapsulate

Y-encapsulated 527 SCE

Y-spray dried 379 SCE

Figure 2. Change in pH (A)* and syneresis (%) (B) of control yoghurt, yoghurt sample containing freeze dried SCE, yoghurt sample containing blank encapsulate, yoghurt sample containing encapsulated SCE, yoghurt sample containing freeze-dried SCE during 21 days of storage at 4°C. *sd < 0.005

100 95

L* value

90 85 80 75 70 1

147 Control Yoghurt

7

Storage period (days): 1-7-14-21 Y-freeze dried SCE 198

Y-blank 642

encapsulate

14

21

527 Y-encapsulated SCE

Y-spray dried SCE 379

92 90 88

WI

86 84 82 80 78 76 74 1

7

14

21

Storage period (days), 1-7-14-21 147 Control Yoghurt

Y-freeze dried 198 SCE

Y-blank 642

Y-encapsulated SCE 527

encapsulate

Y-spray dried SCE 379

14 12

Chroma

10 8 6 4 2 0 1

7

14

21

Storage period (days), 1-7-14-21 Control Yoghurt 147

Y-freeze dried SCE 198

Y-blank 642

encapsulate

Y-encapsulated SCE 527

Y-spray dried SCE 379

80 70 60

Hue

50 40 30 20 10 0 1

7

14

21

Storage period (days), 1-7-14-21 Control Yoghurt 147

Y-freeze dried SCE 198

Y-blank 642

encapsulate

Y-encapsulated 527 SCE

Y-spray dried SCE 379

Figure 3. Change in color parameters of control yoghurt, yoghurt sample containing freezedried SCE, yoghurt sample containing blank encapsulate, yoghurt sample containing encapsulated SCE, yoghurt sample containing spray dried SCE during 21 days of storage at 4°C.

18,00 16,00

TPC (mg GAE/100g)

14,00 12,00 10,00 8,00 6,00 4,00 2,00 0,00

Control Yoghurt

Y-freeze dried SCE

Y-encapsulated SCE

Y-spray dried SCE

Storage period (days):

1

7

14

21

10 9

mg TEAC/100g

8 7 6 5 4 3 2 1 0

Control Yoghurt

Y-freeze dried SCE

Y-encapsulated SCE

Y-spray dried SCE

Storage period (days):

1

7

14

Figure 4. Change in TPC and antioxidant capacity of control yoghurt, yoghurt sample containing freeze dried SCE, yoghurt sample containing liposomal powder with SCE, yoghurt sample containing spray dried SCE during 21 days of storage at 4°C. *Values are presented as mean values ±standard deviation (n=3). Different small letters represent statistically significant differences (p<0.05) from samples at defined days. Different capital letters represnet statistically significant differences (p<0.05) between storage days for each sample.

21

Highlights >Yoghurt was employed as a vehicle for inclusion of liposomal powder with phenolics.>Spray drying did not degrade phenolic compounds encapsulated in secondary liposomes.>Liposomal encapsulation provided stability for sour cherry extract during storage..>Liposomal powder addition reduced syneresis, but not approved by sensory analysis.> Sensorial properties of all yoghurts were similar such as gel firmness, color, etc.

This research has not been submitted for publication nor hasit been published in whole or in part elsewhere. We attest to the fact that all Authors listed on the title page have contributed significantly to the work, have read the manuscript, attest to the validity and legitimacy of the data and its interpretation, and agree to its submission to the Food Hydrocolloids. Corresponding Author: Prof. Dr. Beraat Ozcelik