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)
28
were converted into powder form by spray drying to make the use of such delivery systems
29
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
52
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
54
advantage of liposomal encapsulation is to provide high loading capacity for partially or
55
completely water-soluble components. Thus, the liposomal systems offer a better delivery
56
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
59
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
62
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
65
with the cocoa phenolics incorporated by aqueous liposomal dispersion in terms of total
66
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
72
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
86
with higher antioxidant activity. Similarly, Ghorbanzade et al. (2017) fortified yoghurt
87
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
89
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
94
(uncoated) and secondary (coated) liposomes, and liposomal powder. Scanning Electron
95
Microscope (SEM) images were also taken for liposomal powder. The aims of this study were
96
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
98
liposomal systems are applicable to a food system (stirred-type yoghurt) in terms of sensorial
99
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
101
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,
112
glacial acetic acid, sodium acetate trihydrate, and Folin-Ciocalteu reagent were purchased
113
from Merck KGaA (Merck, Darmstadt, Germany). 20 DE value of Maltodextrin (MD) was
114
provided from Tunckaya Kimyevi Maddeler Ticaret ve Sanayi Inc., Turkey. Sephadex G-50
115
was supplied from GE Healthcare Life Sciences (Uppsala, Sweeden).
116
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
118
stirred for 15 min for 3 times. Methanol was removed under reduced pressure using a rotary
119
evaporator (Bibby Sterilin RE-100, Bibby Scientific Limited, Staffordshire, UK) at 40 °C
120
until the acetone had been removed. The remaining aqueous extract was collected and freeze
121
dried (Alpha 1-2 LDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz,
122
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,
125
freeze dried sour cherry extract (SCE) (0.2%, w/w) was dissolved in lecithin solution. For
126
homogenization of the lecithin dispersion, a high shear disperser (DI-25 Yellowline, IKA)
127
was employed for 10 min at 9500 rpm. A high pressure homogenizer (Microfluidizer
128
Processor M-110L, Microfluidics, Newton, USA) was operated at homogenization pressure of
129
25,000 psi to obtain primary liposomes without SCE and with SCE (0.2%, w/w). Lecithin
130
solutions with and without SCE were passed five times through a high pressure homogenizer.
131
The chamber of homogenizer was cooled during the homogenization with ice water to prevent
132
the heating of samples. Secondary liposomes were produced via layer-by-layer deposition
133
method. For this aim, liposomes with and without SCE were added to chitosan solutions
134
(0.4%, w/v) in acetate buffer solution (pH = 3.5 ± 0.1; 0.1 M) stirred overnight at room
135
temperature. Therefore, the surface of negatively charged primary liposomes was covered
136
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
139
% (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
141
blank, SCE (0.1%, w/w) was dissolved in acetate buffer, mixed with MD (1:1) (40% w/v in
142
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
147
temperature, 90 °C at outlet temperature and 0.67 m3/min air flow. Collected dried powders
6
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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
159
particle size and ζ-potential were analyzed using the above mentioned method.
160
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
164
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%
168
(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
174
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
183
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
187
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
195
min at 4000 rpm at 20 °C. The supernatant was removed and the centrifuge tube containing
196
sediment was weighed. All analyses were performed in triplicates. The water absorption
197
capacity was determined by the below equation:
(1)
∗
(2) .
(3)
8
198
(
)=
× 100
is the weight of yogurt (g),
(4)
199
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)
203
to determine the stability of encapsulated SCE in yoghurt formulation according to the
204
method of de Carvalho et al. (2018). Briefly, 20 g of yoghurt sample was treated with 30 ml
205
of acidified acetone solution, was vortexed, and kept at + 4°C overnight. After filtrating
206
through Whatman No:2 filter paper, acetone was evaporated from the collected aqueous phase
207
at 40 oC by using rotary evaporator. Following centrifugation at 10,000 rpm for 2 min,
208
collected top-aqueous phase was freeze dried and kept at -80 °C.
209
2.12 Determination of total phenolic content
210
The total phenolic content (TPC) was measured by the Folin-Ciocalteu assay according to the
211
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),
213
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
223
respectively. After keeping in dark for 25 min, the mixture was centrifuged at 300 rpm for 5
224
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
231
direction of the panel leader to develop a sensory language and describe the sensory
232
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
234
separation. The degrees of sensory quality scores were between 1 and 5 for each category,
235
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
240
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
246
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
248
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
252
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
256
liposomes with SCE (0.1%, w/w) were mixed with MD (40%, w/w) and then spray dried. As
257
seen in Table 1, the particle size of reconstituted samples were similar or slightly smaller than
258
before spray drying (p > 0.05) owing to the potential reducing water activity of salts and
259
sugars. The concentration gradient between the inside and outside of the liposomes might
260
change after the addition of MD. The water in the core of liposome may migrate after mixing
261
with the MD, hence the concentration gradient was decreased. Thus, the reduction of particle
262
size may be explained by the osmotic driving force (Karadag et al., 2013). In addition,
263
moisture content of samples was 73.89 ± 2.06, 75.92 ± 2.35, and 76.71 ± 1.89 for liposomal
264
powder without and with SCE, and SCE powder, respectively. There was no significant
265
difference between the detected samples (p > 0.05) in terms of moisture content.
266
3.2 Content and location of SCE in primary and secondary liposomes, and liposomal
267
powder
268
Location of phenolics can be the interior of liposomes, partially in the phospholipid
269
membrane or onto the surface of liposomes. The primary liposomes containing 0.1% (w/w)
270
extract showed the highest encapsulation efficiency (71.2%), which means that 71.2% of the
271
SCE was attached to the surface and the interior of the primary liposomes. Our results are
272
comparable with the results reported before by Gibis, Vogt & Weiss (2012) (83.5% for grape
11
273
seed extract 0.1%, w/w). In this study, 12.81 mg/L of phenolic substances were detected on
274
the surface of the liposomes after gel filtration. In order to calculate the phenolic content
275
inside the liposomes, the concentration detected after gel filtration was subtracted from the
276
concentration detected after Triton treatment of filtered samples. This yielded concentration
277
of 14.41 mg/L. The efficiency of phenolic substances incorporation in secondary liposomes
278
with SCE was found at 78.5%. In this case, the phenolic content on the surface was detected
279
only 9.5 mg/L and the content in the interior of the liposomes was determined that 15.31
280
mg/L. This indicates that coating the liposome surface with chitosan polymer makes the
281
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
283
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
285
accessibility of phenolics inside or on the surface of primary liposomes (Gibis, Vogt & Weiss,
286
2012). After spray drying of secondary liposomes with SCE, the total amount of detectable
287
phenolics was found that 37.52 ± 1.13 mg/L. Compared to the results of TPC of liposomal
288
dispersions (38.19 mg/L) before spray drying, it was observed that spray drying process did
289
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
291
degrade phenolic compounds encapsulated in liposomes due to the protective function of both
292
the liposomal membrane and the biopolymer coat.
293
3.3 Powder morphology of liposomal powders
294
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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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