Comparative study of instrumental properties and sensory profiling of low-calorie chocolate containing hydrophobically modified inulin. Part 1: Rheological, thermal, structural and external preference mapping

Journal Pre-proof Comparative study of instrumental properties and sensory profiling of low-calorie chocolate containing hydrophobically modified inulin. Part 1: Rheological, thermal, structural and external preference mapping Maryam Kiumarsi, Dorota Majchrzak, Samira Yeganehzad, Henry Jäger, Mahdiyar Shahbazi PII:

S0268-005X(19)32062-4

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105698

Reference:

FOOHYD 105698

To appear in:

Food Hydrocolloids

Received Date: 6 September 2019 Revised Date:

14 January 2020

Accepted Date: 21 January 2020

Please cite this article as: Kiumarsi, M., Majchrzak, D., Yeganehzad, S., Jäger, H., Shahbazi, M., Comparative study of instrumental properties and sensory profiling of low-calorie chocolate containing hydrophobically modified inulin. Part 1: Rheological, thermal, structural and external preference mapping, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105698. 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. © 2020 Published by Elsevier Ltd.

Maryam Kiumarsi: Collecting data, Data interpretation, Methodology, Modelling, and Writing – Original draft. Dorota Majchrzak: Investigation, Data Collection, Supervision, and Review & Editing. Samira Yeganehzad: Review & Editing. Henry Jäger: Review & Editing. Mahdiyar Shahbazi: Conceptualization, Methodology investigation, Validation, Data interpretation, Funding acquisition, Writing – Original draft, Writing – Review & Editing, and Supervision the entire study.

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Comparative study of instrumental properties and sensory profiling of low-calorie chocolate

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containing hydrophobically modified inulin. Part 1: Rheological, thermal, structural and external

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preference mapping

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Maryam Kiumarsia, Dorota Majchrzaka, Samira Yeganehzadb, Henry Jägerc, Mahdiyar Shahbazic* of Vienna, Department of Nutritional Sciences, Faculty of Life Sciences, Althanstraβe 14, A-1090 Vienna, Austria bResearch Institute of Food Science and Technology (RIFST), PO Box 91735-147, Mashhad, Iran cInstitute of Food Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austria aUniversity

ABSTRACT

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Low-calorie chocolate was prepared using replacement of sucrose by hydrophobically modified inulin

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(dodecenyl succinylated inulin) as biopolymeric surfactant at different levels (100:0, 75:25, 50:50, 25:75,

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and 0:100%). The instrumental parameters of produced chocolates before and after storage were

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compared with their sensory evaluation. Morphological assay showed that the lowest modified inulin ratio

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allowed an increase in frequency of surface crystals upon storage with progress of blooming phenomenon.

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However, higher levels of modified inulin were more effective in enduring blooming. Replacement of lowest

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inulin ratio increased elastic modulus of stored chocolate, with a more solid-like behavior. However,

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viscoelastic parameters of stored chocolates with higher inulin contents remained similar to the levels

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obtained for non-stored samples. Thermal analysis revealed that enthalpy increased in stored chocolate

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containing lowest inulin content due to post crystallization, while substitution of inulin at higher levels

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seemed to slow down this process. Upon storage, V-type crystal was transformed to VI-type form in

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chocolate formulated with the lowest modified inulin proportion, while chocolates with higher inulin contents

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showed the slowest change in polymorphic transformation. Quantitative descriptive analysis revealed that

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increasing inulin content resulted in good textural and color appearance. Partial least squares showed that

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blooming, mass forming, cracking and powderiness attributes were responsible for lower consumer

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acceptance of chocolate.

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Keywords: Low-calorie chocolate; Biopolymeric surfactant; Phase separation; Strain sweep; XRD;

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Sensory evaluation.

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Graphical Abstract

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1. Introduction

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Chocolate is a high energy product with a unique taste and texture, composed by sugar and fat in high

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proportion, as the main sources of energy. Increasingly, consumers and manufacturers are becoming

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concerned about the high-caloric components and the carcinogenicity of ingredient included in chocolate

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products, therefore popularity of ‘light’ and ‘low-calorie varieties is growing (Aidoo, Depypere, Afoakwa, &

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Dewettinck, 2013; Shah, Jones, & Vasiljevic, 2010). In recent years, many efforts have been promoted to

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replace the most common sweetener used in chocolate formulation, namely sucrose, to produce a low-

38

calorie and healthier food (Nebesny, Żyżelewicz, Motyl, & Libudzisz, 2007; Aidoo et al., 2013). Sugar offers

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multi-functional properties as sweetener, bulking agent and provides textural characteristics to chocolate

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products. Production of low-calorie chocolate is most challenging since partial or whole amount of sugar

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needs to be replaced. Moreover, sugar replacement by bulk sweeteners affects the functional quality

42

characteristics like rheological and textural properties, melting behaviors, bloom formation and other

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parameters that influence the final stability of product. Therefore, some innovative strategies need to be

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developed and implemented for chocolate formulation. It is shown that the incorporation of bulk

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sweeteners, fiber or fiber-like ingredients to chocolate formulation can provide suitable physical, rheological

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and textural characteristics to achieve an acceptable low-calorie product (Nebesny et al., 2007; Meyer,

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Bayarri, Tárrega, & Costell, 2011; Aidoo et al., 2013).

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Inulin is a natural fructan constituted by β(2,1)-linked fructosyl residues, ending with a glucose residue and

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it is present as storage carbohydrate in a large number of plants (Meyer et al., 2011; Kokubun, Ratcliffe, &

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Williams, 2015). Inulin provides many functional benefits along with certain industrial properties not only

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due to its gelling features, but for its nutritional and health-related benefits as dietary fiber (Roberfroid,

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2005). The technological use of inulin in chocolate is based on its properties as sugar replacer and texture

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modifier (Meyer et al., 2011). Inulin as a sugar substitute is generally capable to mimic the sucrose

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functional properties such as mouthfeel and texture in low-calorie chocolates. The applicability and 3

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suitability of inulin as a bulk sweetener in low-calorie chocolate have been studied. Konar et al. (2018)

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investigated the inulin effect with different polymerization degree on quality parameters of sugared and

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sugar-free chocolates and reported that inulin with high polymerization degree had significant effects on

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melting point, water activity, textural properties and color parameters. Konar, Özhan, Artık, Dalabasmaz,

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and Poyrazoglu. (2014) also evaluated the effects of inulin at different concentrations on functional

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properties of milk chocolate and found that hardness, water activity, yield stress and viscosity changed after

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inulin addition. Shah et al. (2010) replaced sucrose with inulin at different degrees of polymerization and

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polydextrose as bulking agents. The melting point temperature of chocolate formulated with inulin showed a

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considerably higher value compared to the other samples. Golob, Micovic, Bertoncelj, and Jamnik (2004)

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studied the influence of inulin on sensory characteristics of chocolate by a consumer panel. They reported

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that chocolate formulated with use of inulin as sucrose replacer did not result in sensory differences

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compared to control sample.

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In recent years, the use of biopolymeric surfactants i.e., high molecular weight surfactants, in fat-based

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food suspensions as emulsifiers and stabilizers has attracted much attention (Do, Mitchell, Wolf, & Vieira,

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2010; Ceballos et al., 2016). Surfactants are an important ingredient in the production of chocolate,

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because can coat the surfaces of sugar and cocoa particles dispersed in fat phase, generally cocoa butter,

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to preserve or improve the flowability of molten chocolate (Rodriguez Furlán, Baracco, Lecot, Zaritzky, &

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Campderrós, 2017). Coating the surfaces of solid particles dispersed in chocolate with biopolymeric

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surfactant reduces the inter-particle interactions responsible for particle aggregation, which can contribute

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to effective dispersion stability in a different manner compared to low molecular weight surfactants (Do et

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al., 2010). The appropriate biopolymers for the stabilization of fat-based suspensions, like chocolate, must

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offer adequate interfacial activity and should be soluble in the continuous lipid phase. Recently, inulin has

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been widely modified with chemical and enzymatic approaches to produce fatty acid ester derivatives,

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which have potential application as biopolymeric surfactants to stabilize dispersions and emulsions 4

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(Kokubun et al., 2015). It has been shown that the hydrophobically modification process of inulin can be

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readily achieved by its interaction with alkenyl succinic anhydride in aqueous solution and obtained

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products are surface-active and can efficiently stabilize fat-based suspensions (Kokubun et al., 2015).

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Hydrophobically modified inulins are employed commercially as an alternative of purely polymeric

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surfactants used in many colloidal systems as stabilizers of solid dispersed in a liquid phase (Tadros,

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Vandamme, Booten, Levecke, & Stevens, 2004). Emulsions and suspensions prepared using

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hydrophobically modified inulin show a number of interesting features when compared with other

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surfactants. Firstly, stable dispersions can be obtained with much lower biopolymeric surfactant

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concentration. Secondly, these dispersions are very stable in high electrolyte concentrations and high

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temperatures. Moreover, these dispersions and emulsions are free from any strong (irreversible)

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flocculation and coalescence (Tadros et al., 2004).

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Despite the amphiphilic nature of hydrophobically modified inulin, it has not been studied as a biopolymeric

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surfactant for the stabilization of solid particles in the real fat-based food suspensions (like chocolate

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systems). Moreover, the applicability and suitability of hydrophobically modified inulin as sucrose replacers

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and biopolymeric surfactant during manufacture of low-calorie chocolate are yet to be fully understood.

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Development of a high-quality low-calorie chocolate needs the use of the most appropriate ingredients that

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could substitute sugar without negatively affecting the final functional properties of product. In the present

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study, sucrose was replaced with dodecenyl succinylated inulin in chocolate formulation and changes in

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functional features of non-stored and stored chocolates were investigated in relation to rheological

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properties, thermal behavior, crystalline pattern and morphological characteristic, as well as sensory

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evaluation. Relationship between the descriptive sensory attributes and the acceptance test was evaluated

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by partial least squares regression model. Since there is no published report in the area of chocolate

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formulated with hydrophobically modified inulin, the results of the current study were compared with those

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from studies that used non-modified inulin. 5

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2. Materials and methods 2.1. Materials

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Cocoa butter and cocoa powder were obtained from Guan Chong Cocoa Manufacture SDN (BHD,

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Malaysia). The cocoa powder was lightly alkalized dark brown with pH in the range of 6.8–7.1. The

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moisture and fat content of cocoa powder were 3.42 wt.% and 13.66 wt.%, respectively. Soy lecithin (Lucas

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Meyer GmbH, Hamburg, Germany) had a moisture content of 0.11 wt.% and a value of 64.25 wt.% for

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acetone insolubles. Sugar powder was obtained from Sari Nira Nusantara CV (Yogyakarta, Indonesia).

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Inulin (FXL, Cosucra, Belgium) as a biopolymeric surfactant or stability agent with an average DP > 23 was

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modified by (2-dodecen-1-yl)succinic anhydride (Kokubun et al., 2015; Kiumarsi et al., 2019). The obtained

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modified inulin was dried in air dry oven (Heraeus, Thermo Fisher Scientific, Munich, Germany) at 105 °C

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for 18 h, and then filtered using a sieve with a mesh size of 74 µm to reach the proper particle size. For

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inulin modification, triplicate analyses for two samples from two separate batches of product were tested

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(Kiumarsi et al., 2019). The changes in the chemical bonds and functional groups of inulin after

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hydrophobically modification treatment were indicated by FT-IR spectroscopy (Fig. A-1 in Supplementary

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Material). For interpretation of the reference and preparation of native inulin and dodecenyl succinylated

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inulin in this section, the reader is referred to the Supplementary Material of this article.

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2.2. Chocolate preparation

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Five batches (3 kg each) of chocolate were prepared using the same procedure with following ingredients.

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Sucrose (25.7 wt.%) was replaced with different levels of hydrophobically modified inulin (100:0, 75:25,

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50:50, 25:75 and 0:100%) in chocolate formulation. Other ingredients including cocoa butter (44.5 wt.%),

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cocoa powder (29.7 wt.%) and soy lecithin (0.1 wt.%) were weighed and mixed in Vema mixer (Vema BM

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30/20, Vemacon-struct, NV Machinery Verhoest, Izegem, Belgium) at temperature of 45 °C and rotational

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speed of 5 rpm for 60 min. Chocolate samples were prepared in semi-industry conditions with using a 3-

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roll refiner (Buhler Ltd., Uzwil, Switzerland) to a specified particle size of 25-28 µm in batches of 3 kg per 6

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formulation. The refined chocolates were conched (IMC-E10, Mannhein, Germany) at 48 °C for 12 h and

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finally tempered using Little Dipper Tempering Machine (Hilliards Chocolate System, West Bridgewater,

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MA, USA). The tempered mass was molded into polycarbonate molds and allowed to cool in refrigerator

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(4 °C) for 30 min before de-molding. The finished bars were wrapped in aluminum foil and stored at 18 °C

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for later analysis. The codes of MI-25, MI-50, MI-75 and MI-100 were considered for the samples with

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sucrose to modified inulin proportion of 75:25, 50:50, 25:75, and 0:100%, respectively.

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2.3. Flow curve

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Flow properties of chocolate as affected by inulin substitution and storage condition were investigated by

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rheometer (AR 2000, TA Instruments, New Castle, DE, USA) with using concentric cylinder system (cup

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and bob) (Aidoo et al., 2013). First, chocolate variants were heated by air dry oven (Heraeus, Thermo

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Fisher Scientific, Munich, Germany) at temperature of 50 °C for 60 min to melt. Then, molten chocolate

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samples (10 g) were weighed into the cup and measurements were performed by the ICA (2000). Samples

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were pre-sheared at shear of 5 s−1 at 40 °C for 10 min before starting the measurement cycle. Tests were

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conducted at 40 ºC using parallel plate geometry, with 40 mm diameter. Shear stress ( ) was measured as

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a function of increasing shear rate ( ) from 2 s−1 to 100 s−1 (ramp up), holding at 100 s−1 for 60 s, then

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decreasing from 100 s−1 to 2 s−1 (ramp down). The flow curve was then obtained by plotting the recorded

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shear stress as a function of the applied shear rate. The best equation was selected by statistical analysis

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and the rheological parameters were calculated using the best model. In this regard, plastic viscosity, flow

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behavior index and yield stress values were obtained by fitting the Herschel-Bulkley model (Eq. 1) to the

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data. The effectiveness of Herschel-Bulkley equation was verified using statistical analysis, by residual

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plots and normally test through statistical software of Graph Pad In Stat (Sokmen & Gunes, 2006).

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=

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where

Eq. (1)

+ is the yield stress, K is the plastic viscosity, n is the flow behavior index.

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2.4. Oscillatory tests

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In dynamic conditions, oscillatory measurements by using rheometer (AR 2000, TA Instruments, New

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Castle, DE, USA) were conducted with plate–plate geometry in order to explore the viscoelastic parameters

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of chocolate variants and to evaluate the storage (G′) and loss (G″) moduli at a temperature of 40 °C.

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Aliquots of samples (about 4–5 g) were transferred on the temperature-controlled measuring plate and the

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measuring gap was set at 1000 µm. These processes were conducted to prevent any possible damage of

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the crystalline network. In order to detect the linear viscoelastic range (LVR), strain sweep tests were

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applied. Rheograms were also determined by plotting the complex modulus (G*) as a function of oscillatory

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shear. The complex modulus can be showed as the ratio of stress over the relevant strain and is

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considered to be an index of the stiffness of the system. The end of the LVR was marked by the first point

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where the G′ varies by 10% of the G′ in the LVR, and the corresponding stress at this point was referred to

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as the critical oscillation stress (ICA, 2000).

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2.5. Differential scanning calorimeter

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The thermal behaviors of chocolate as affected by substitution of modified inulin and storage time were

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evaluated by a differential scanning calorimeter (DSC-Q100, TA Instruments, New Castle, DE, USA). The

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DSC instrument was calibrated by lead (332.5 °C), tin (216.7 °C), indium (138.8 °C) and distilled water.

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The chocolate samples (10 mg) were transferred to an aluminum pan and heated from 0 °C to 240 °C with

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a heating rate of 10 °C.min-1 under an oxygen free nitrogen flow rate of 50 mL.min-1. Peak onset (To)

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corresponds to the temperature at which a specific crystal form starts to melt; peak maximum (Tp) assigns

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to the temperature at which melting is greatest, and end of melting (Te) relates to completion of liquefaction

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(Shahbazi, Rajabzadeh, & Sotoodeh, 2017). These parameters were automatically calculated after

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integrating the melting peaks through TA Data analysis software (TA Instruments, New Castle, USA). All

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these information are related to crystal type (Shahbazi, Majzoobi, & Farahnaky, 2018a).

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2.6. X-ray diffraction patterns (XRD) of chocolates

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Powder XRD patterns and relative crystallinity of the chocolate variants were recorded by Rigaku D/Max-b

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X-ray diffractometer (Rigaku Corp., Tokyo, Japan) in order to identify the polymorphic transformations of

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chocolate. The test was performed with 40 kV energy, 30 mA current and Cu Kα irradiation (λ= 1.54056 Å)

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at 18 °C (AOCS 2009; Shahbazi, Rajabzadeh, Rafe, Ettelaie, & Ahmadi, 2017). The samples were

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irradiated in the range of 1-50° and scanned with a speed of 0.018°.min-1. XRD is measured by Bragg’s

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law: nλ = 2dsinθ; where n is a positive whole number, λ is the X-ray wavelength, d = space between crystal

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planes and θ is the angle of incidence. To determine the relative crystallinity (RC) of chocolates, total curve

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area (At) and the area under the XRD peaks (Ap) were measured by the software developed by the

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manufacturer (EVA, Version 9.0) and the relative crystallinity was obtained using Eq. 2 (Shahbazi,

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Majzoobi, & Farahnaky, 2018b): RC (%) = (A ⁄A ) × 100

184

Eq. (2)

2.7. Morphology structure of chocolates

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The influences of modified inulin replacement and storage time on morphological structure of the chocolate

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samples were observed through a variable-pressure scanning electron microscope (VP-SEM Quanta 200

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FEG SEM, FEI Company, Eindhoven, Netherlands) to produce high-resolution images with a high depth of

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field. Initially, the samples were cut into the size of (10 × 10 × 10) mm3. To avoid thermal damage, the

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sample was mounted on a peltier-cooled stage with the temperature set to −5 °C (James & Smith, 2009),

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and nitrous oxide was used as an imaging gas with a pressure of 50.7 Pa. The distribution of particles in

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each sample was imaged through a solid state backscatter detector and an accelerating voltage of 20 kV

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(Majzoobi, Shahbazi, Farahnaky, Rezvani, & Schleining, 2013).

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2.8. Sensory evaluation 2.8.1. Quantitative descriptive analysis (QDA)

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The QDA evaluation was carried out according to Lawless and Heymann (2010) using ten trained panelists

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(5 males and 5 females, aged 20–35 years) preselected out of 20 with a triangle difference test regarding to

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their discriminating capacity (P ≤ 0.30), reproducibility capacity (reliability) (P > 0.05) and individual

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consensus during training sessions. All these panelists were completely familiar with sensory evaluation of

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different kinds of chocolate products as a result of participating in several QDA investigations (see section

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A-2 in Supplementary Materials). The preselected assessors were rigorously trained for the QDA

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evaluation according to the guidelines of the ISO 8586:2012 standard (ISO, 2012). During the first

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discussion session, the panelists generated and agreed upon 21 attributes terms with definitions, which

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well described the chocolate samples characteristics (Table A-1 in Supplementary Materials). References

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were used, where needed, to clarify and better understanding of the terms.

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Sensory evaluations using QDA were performed at the sensory laboratory designed in accordance with

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ISO 8589:2007/Amd.1:2014 at the Department of Nutritional Sciences (Vienna, Austria) in individually air-

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conditioned booths equipped with computers under the normal lightening condition at room temperature.

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Ten chocolate variants (five non-stored and five stored) with the same size and weight were coded with

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three-digit numbers and served in randomized order using a complete block design to avoid artifacts owing

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to order of sample presentation (Lawless & Heymann, 2010). Water was provided to drink between each

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sample evaluation for palate cleansing.

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The panelists received the samples and were asked to rate the intensity of each attribute, using a

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continuous 10-cm unstructured line scale anchored on the left by “weak” or “none” and on the right by

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“strong” or “much.” The samples were evaluated in 3 repetitions (each repetition was performed in one

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session) and finally, QDA data were collected using FIZZ software (Biosystèmes, v2.51a 86, Couternon,

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France). 10

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2.8.2. Acceptance test

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Acceptance test was performed according to Lawless and Heymann (2010) in order to evaluate the overall

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liking of chocolate samples with 9-point hedonic scale, ranging from 1 (dislike extremely) to 9 (like

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extremely) using FIZZ software. It was carried out with 120 consumers consisting, 50 male and 70 female.

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The panelists were aged between 18 and 52 years old. All participants were consumers of chocolate (at

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least 3 times a week). The chocolate variants were presented with a three-digit number and evaluated over

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one session in individual air-conditioned booths under normal light illumination. The panelists were

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instructed to rinse their mouths with water between samples. In order to avoid any fatigue, no added

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information about the samples was given to the consumers.

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2.9. Statistical analysis

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All experiments were performed as triplicate determinations and the mean and standard deviation of the

228

data were reported. Analysis of variance (ANOVA) was applied for the determination of the main effects of

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the investigated independent factors (ratio of modified inulin to sugar) and their interactions on the

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instrumental and sensory data. Duncan’s multiple range test was used to separate means of data when

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significant differences (P < 0.05) were observed.

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Linear correlation (Pearson’s correlation coefficients) was applied on the instrumental and sensory data to

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reveal their particular interrelationships with XLSTAT software (Addinsoft SARL, New York, NY, USA).

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Descriptive information obtained from the trained panel was related to the consumer acceptance data using

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partial least squares regression (PLS). The overall impression was the dependent variable (Y-matrix) while

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the QDA descriptive terms were the independent variables (X-matrix) (Cadena et al., 2013).

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3. Results and discussion

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3.1. Flow curve

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The flow behavior of chocolate is an important functional characteristic directly related with an optimal

240

mouthfeel (Rodriguez Furlán et al., 2017). Fig. 1 presents the flow curves of the different essayed 11

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chocolate formulations as a function of sugar replacement by modified inulin. The results indicated that the

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chocolates flow with a typical non-Newtonian behavior (De Graef, Depypere, Minnaert, Dewettinck, 2011).

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In order to better explain the rheological values obtained by the flow curves, Table 1 shows the rheological

244

parameters for different samples, fitted to the Herschel-Bulkley equation, providing high correlation

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coefficients (R2) varied from 0.96 to 0.99. The diagnostic analysis of the Herschel-Bulkley equation

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revealed residual plots with no systematic patterns and generally distributed with P > 0.1 for all chocolate

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samples presenting a Gaussian distribution (R2 ≈ 1) (Rodriguez Furlán et al., 2017).

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Table 1. The summary of plastic viscosity, flow behavior index and yield stress obtained for different chocolate samples. Samples

Yield stress (Pa)

Plastic viscosity (Pa.sn)

R2

Flow behavior

Non-stored

Stored

Non-stored

Stored

Non-stored

Stored

Non-stored

Stored

Control

6.76±0.21a

32.06±0.20c

1.77±0.06a

3.35±0.02b

0.69±0.022a

0.72±0.013a

0.975

0.995

MI-25

7.57±0.22b

34.66±0.24d

1.78±0.04a

3.18±0.03a

0.74±0.014b

0.74±0.011b

0.986

0.985

MI-50

10.38±0.19c

10.72±0.30a

2.02±0.06b

2.05±0.07c

0.77±0.011c

0.76±0.008c

0.985

0.962

MI-75

10.91±0.28d

11.01±0.26a

2.07±0.04b

2.09±0.05c

0.79±0.012d

0.78±0.011d

0.977

0.985

MI-100

11.56±0.24e

11.97±0.25b

2.17±0.05c

2.20±0.03d

0.78±0.020cd

0.78±0.010d

0.985

0.973

249

a–e Means (three replicates) within each column with different letters are significantly different (P < 0 .05), Duncan’s test.

250

Generally, sucrose replacement with modified inulin resulted in higher flow behavior index (P < 0.05) and

251

there was no significant difference among the stored and non-stored samples (P > 0.05). This is in

252

agreement with previously reported finding regarding the effect of inulin on the rheological properties of

253

chocolate (Shah et al., 2010). A flow behavior index lower than 1 indicates slight shear-thinning behavior

254

above the yield stresses. As summarized in Table 1, the non-stored chocolates formulated with modified

255

inulin had a higher flow index than non-stored control with a pseudoplastic behavior (0.69 < n < 0.79),

256

similar to the study performed by Sokmen and Gunes (2006) on low-calorie chocolate. It will be shown later

257

that inulin-added chocolates exhibited higher relative crystallinity degree values (section 3.5); hence, the

258

added modified inulin is probably the cause of such higher flow behavior responsible for the shear-thinning

259

effect displayed by the chocolates. Presence of more crystals in the chocolates with modified inulin could 12

260

have caused difficulty in crystal alignment during the chocolate manufacturing process, which resulted in an

261

increase in the flow behavior index (Aidoo et al., 2013). Shah et al. (2010) reported that sucrose

262

replacement with inulin in chocolate resulted in higher flow behavior index. Table 1 also shows that different

263

amounts of modified inulin had no significant effect on the flow behavior of stored chocolates compared to

264

their non-stored samples (P > 0.05).

265

Fig. 1. Flow curves of non-stored (left) and stored chocolates (right) as a function of modified inulin substitution.

266

The plastic viscosity, which is related to the chocolate consistency (De Graef et al., 2011), showed an

267

increase when modified inulin replaced sucrose at a range of 50% to 100% (Table 1). As summarized in

268

Table 1, non-stored MI-100 presents the highest value of viscosity with initial value of 2.17 Pa.s, followed

269

by MI-75 with viscosity value of 2.07 Pa.s and MI-50 with value of 2.02 Pa.s. Higher viscosity value with

270

modified inulin might be associated with higher solid volume fraction in the chocolate, which increases the

271

particle–particle interactions, decreasing their mobility that involved an increase of viscosity (Aidoo et al.,

272

2013). These results are supported by the studies of Shah et al. (2010) and Aidoo et al. (2013) that noticed

273

a higher viscosity for inulin-containing chocolates compared to control sample. In the light of this

274

information, the chocolate containing highest level of modified inulin (MI-100) might cause a stronger pasty

275

mouth feeling when consumed in comparison with the other samples (Beckett, 2011). 13

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In the case of stored chocolates, changes in the viscosity did not follow a special trend. The viscosity

277

values of MI-50 and MI-75 were almost unaffected upon storage compared to their non-stored samples,

278

which indicated no change in the rheological properties after three months of storage (Table 1). In contrast,

279

viscosity values of control and MI-25 samples were increased after storage. Viscosity of stored chocolate is

280

closely connected with amount of fat immobilized on the particle surface. This suggests that

281

hydrophobically modified inulin at the intermediate levels (50% and 75%), as biopolymeric surfactant, could

282

effectively coat the solid particles, preventing the displacement of the particles upon storage. Moreover, in

283

the presence of modified inulin (the intermediate levels) sugar particles were more efficiently dispersed and

284

less aggregated (Do et al., 2010). The improved particle dispersion in the stored MI-50 and MI-75 proposed

285

that modified inulin co-adsorbs at the sugar surface together with lecithin, forming a mixed interfacial film

286

with the lecithin at the oil–water interface (Do et al., 2010).

287

Yield stress is important in keeping small solid particles in fat-based suspensions and in the coating of solid

288

surfaces. From Table 1, it is clear that an increase in the ratio of modified inulin to sucrose affects the yield

289

values of non-stored samples. Compared to non-stored control, replacement of modified inulin at the lowest

290

level (MI-25 sample) caused a slight increase in yield stress, while 50% and higher modified inulin

291

constitution resulted in a considerable increase in this parameter. Rodriguez Furlán et al. (2017) reported

292

that inulin at the low content did not change yield stress of chocolate, whereas further increase in added

293

inulin led to an important increase in yield value. Similar results have been reported by Shah et el. (2010)

294

and Aidoo et al. (2013), who described that yield stress of chocolates formulated with inulin were higher

295

than control sample. In the current study, the replacement of modified inulin at the higher concentrations

296

(50-100%) did not induce any change in the yield stress of chocolate upon storage with respect to their

297

non-stored samples, while stored control and MI-25 exhibited a substantial increase in the yield stress

298

values (Table 1). Hydrophobically modified inulin (as biopolymeric surfactant) contribute to the stability of

299

fat-based suspensions, improving the flow properties of the product in time; as it is very effective in the term 14

300

of steric stabilization owing to its molecular size and the formation of multiple binding sites at the interface

301

(Do et al., 2010). This result is important, because the yield stress maintains the small solid particles in the

302

suspension, giving greater stability to the storage chocolate (Sokmen & Gunes, 2006).

303

3.2. Viscoelastic behavior

304

Results of strain sweep test in the terms of storage and loss moduli are shown in Fig. 2. In general, all tests

305

present the similar trend for G′ and G″ and can be divided into three specific regimes. At the small strain

306

amplitude (strain between 0.1-1), the linear viscoelastic regime (LVR) can be seen, where the storage

307

modulus values (G′) are higher than loss modulus (G″) for all samples. This indicates that all chocolate

308

samples behave as a solid with elastic-like properties, suggesting that under non-destructive conditions, the

309

elasticity parameter has a predominant effect on viscosity (De Graef et al., 2011). As visualized in Fig. 2,

310

upon yielding and after LVR (entering the non-linear region), both viscoelastic parameters decrease,

311

suggesting a shear-thinning behavior, where the viscous modulus becomes larger than the elastic one. At

312

the higher strain amplitude, a specific region can be seen, where the moduli increase again. Same result

313

was previously reported by van der Vaart et al. (2013), where dark chocolate showed a complex non-linear

314

behavior, namely shear thinning, shear thickening, and strain stiffening. In the present study, the lower

315

values of G′ were found for non-stored control and MI-25 samples, constituted by a weakly structured

316

system. In contrast, non-stored MI-75 and MI-100 showed the higher values of G′ and G″, while MI-50

317

showed the viscoelastic properties with the intermediate value. In agreement with this behavior, the non-

318

stored MI-100 and MI-25 presented the highest and lowest yield stress, respectively, among the inulin-

319

added samples. It is shown that the level and type of inulin affect both crystallization and aggregation

320

processes, and consequently alter the viscoelastic properties of chocolate (Shah et el., 2010). Fig. 2 also

321

shows that with increasing the ratio of modified inulin to sucrose, the length of LVR, in which the

322

viscoelastic properties are independent from the stress conditions, becomes longer (Shah et el., 2010).

323

These results are more evident when the dynamic data are shown in terms of critical oscillation stress and 15

324

complex modulus (G*) values at the end of the LVR (Table 2). As summarized, with increasing the ratio of

325

modified inulin to sucrose, the critical G* of non-stored samples increased. As the critical G* is a measure

326

for the stiffness of the system (De Graef et al., 2011), it could be concluded that the structure of non-stored

327

chocolate strengthened as the amount of modified inulin increases. Consequently, it can be argued that the

328

critical oscillation stress at the end of the LVR is sensitive to the ratio of hydrophobically modified inulin.

329

This effect was also observed in the Herschel-Bulkley yield value.

330

331

Fig. 2. Storage modulus, G′ (solid symbol) and loss modulus, G″ (open symbol) of non-stored (left) and stored samples (right) as

332

a function of strain for chocolate variants: control (●), MI-25 (▲), MI-50 (♦), MI-75 (▼) and MI-100 (■).

333

Viscoelastic parameters as a function of strain amplitude for stored chocolates are also shown in Fig. 2.

334

After three months of storage, the elastic moduli were still higher than loss moduli, indicating elasticity was

335

the prevalent property of stored chocolates. As shown, the elastic values of stored samples formulated with

336

50% and 75% modified inulin were remained at the levels of their non-stored chocolates. In contrast, the

337

elastic moduli of stored MI-25 (curve not shown) and control effectively increased during storage, which led

338

to the formation of a more solid-like behavior system. Table 2 shows the evolution of the critical oscillation

16

339

stress and critical G* of stored chocolates as a function of different ratios of added modified inulin.

340

Compared to non-stored samples, it can be seen that replacement of sugar by modified inulin at the levels

341

of 50% and 75% did not affect the critical stress and critical G*, whilst these parameters increased in the

342

stored control and MI-25. This indicated that these chocolates were harder and more elastic than other

343

stored ones. Thus, it could be expected that intermediate levels (50% and 75%) of hydrophobically

344

modified inulin could have an effect on both formation, as well stabilization of proper chocolate suspension

345

during storage.

346

Table 2. Critical stress and complex modulus values at the end of the LVR obtained for different chocolate samples.

Samples

347 348

Critical G* × (103) (Pa)

Critical oscillation stress (Pa)

Non-stored

Stored

Non-stored

Stored

Control

75.22±1.61a

851.06±5.43d

0.95±0.05a

3.9±0.09d

MI-25

122.31±2.86b

839.65±6.02c

1.7±0.02b

3.4±0.07c

MI-50

403.12±3.13c

409.66±5.33a

2.3±0.06c

2.4±0.06a

MI-75

416.43±3.62d

418.54±6.76a

2.4±0.04c

2.5±0.09a

MI-100

604.22±6.33e

625.55±7.22b

2.9±0.05d

3.0±0.07b

a–e Means (three replicates) within each column with different letters are significantly different (P < 0.05), Duncan’s test.

3.3. Microscopy

349

VP-SEM micrographs (Fig. 3) showed clear variations in the microstructure for the different chocolate

350

formulations. Non-stored chocolate containing 100% sucrose (control) revealed large solid particles with

351

more void spaces between the particles, indicating limited particle–particle interaction. The VP-SEM

352

micrograph also presented larger crystals for non-stored MI-25, albeit with a little bit denser matrix as

353

compared to control sample. In contrast, non-stored MI-50 and MI-75 showed a high solid packing intensity

354

than those of control and MI-25. Likewise, micrograph of non-stored MI-100 revealed smaller crystals with

355

dense matrix and minimal inter-particle spaces. The high solids packing intensity in chocolate formulation

356

including 50-100% modified inulin could have resulted in higher energy needed to initiate flow, hence,

357

higher plastic viscosity and yield stress. This also explains the increase in elastic modulus and critical G* 17

358

with increasing in the ratio of modified inulin, since the dense packing of samples formulated with higher

359

levels of inulin might limit the flow of chocolate (Aidoo et al., 2013).

360

361

Fig. 3. VP-SEM photomicrographs for non-stored (left) and stored chocolates (right) formulated with different ratios of modified

362

inulin.

363

As visualized from VP-SEM photomicrographs, a high frequency of fat crystals (white fragments) was

364

developed in the surface of stored control sample, where there is certainly destabilization and phase

365

separation of the chocolate suspension (Fig. 3). Likewise, the high frequency of fat crystals with several

366

rough surfaces could be observed for stored MI-25, while stored MI-50, MI-75 and MI-100 showed no

367

development of visible surface crystals and remained free of typical fat bloom alterations upon storage.

368

Accordingly, hydrophobically modified inulin at the intermediate and highest levels led to the formation of a

369

more stable system, and dispersions were free from any strong (irreversible) flocculation and coalescence.

370

Modified inulin can therefore not be used at the low level in producing the low-calorie chocolate due to low

371

impact on preventing the fat crystals development and needs to be used at the higher content to reduce the

18

372

fat blooming. Hence, if sucrose is replaced with higher levels of modified inulin, the rapid deterioration of

373

textural properties and non-desirable appearance during storage could be delayed in the chocolate.

374

3.4. Melting properties

375

Cocoa butter can crystallize in different polymorphs as type I-VI, which among them, type V is the most

376

desirable and type VI is the most stable crystal form (Saputro, et al., 2017). DSC thermograms (Fig. 4)

377

showed that sucrose substitution by modified inulin produced changes in melting behavior and crystallinity

378

of chocolate, found in the differences in the important DSC parameters. The thermograms of non-stored

379

samples (except MI-100) exhibited three distinct endothermic behaviors, attributed to the melting of

380

polymorphic form V and polymorphic form VI, as well sucrose melting/degradation, respectively.

381

Fig. 4. DSC-thermograms of non-stored (left) and stored (right) chocolate variants. Heating rate was 10 °C min−1.

382

As can be seen from thermograms, form VI was abundant polymorphism type in non-stored control and MI-

383

25. This could be related to the presence of low-level of soy lecithin used in the manufacture of chocolate.

384

In contrast, form V was most abundant polymorphism type in non-stored chocolates with higher modified

385

inulin ratios (MI-50, MI-75 and MI-100), suggesting that these samples might have better texture

386

characteristics and a more desirable appearance, as well a good resistance to blooming or more stability.

387

In this way, dodecenyl succinylated inulin at the higher levels acted as lubricant or surfactant. Regarding 19

388

polymorphic form V, To, Tp and Te values of non-stored control sample were determined to be 20.4, 31.4

389

and 35.2 °C, respectively. It is reported that the melting point of polymorphic form V is appeared in the

390

range of 32–34 °C (Saputro, et al., 2017). It is also observed from Table 3 that replacement of modified

391

inulin at higher levels (50-100%) significantly increased the peak maximum (Tp) of form V (P < 0.05), while

392

Tp of chocolate with lowest inulin content (25%) was determined to be closer to non-stored control sample

393

without any significant difference (P > 0.05). An increase in the melting point of chocolate has been

394

observed previously with increasing inulin ratio (Shah et al., 2010).

395

Table 3. Melting properties of non-stored and stored chocolate variants with different ratios of modified inulin to sucrose. Samples

Tp (form V) (°C)

∆H (form V) (J/g)

Tp (form VI) (°C)

∆H (form VI) (J/g)

Non-stored

Stored

Non-stored

Stored

Non-stored

Stored

Non-stored

Stored

Control

31.4±0.5a

nd

12.9±0.6a

nd

42.0±0.4a

43.1±0.4a

41.9±1.6a

93.9±3.1a

MI-25

30.8±0.4a

nd

14.3±0.9a

nd

42.2±0.3a

43.8±0.3a

40.3±1.6a

89.3±2.6b

MI-50

32.5±0.3b

33.1±0.5a

53.6±1.2b

51.8±0.8a

48.6±0.9b

47.7±1.3b

15.5±0.8b

14.6±0.9c

MI-75

32.6±0.4b

33.0±0.4b

56.4±0.6c

54.9±1.0b

48.7±0.8b

48.3±1.5b

16.1±0.5b

14.0±0.7c

MI-100

33.5±0.2c

33.3±0.4c

50.9±0.9d

49.6±1.1c

49.2±1.1b

48.8±1.7b

16.8±0.8b

16.5±1.0c

396 397 398

a–d Means (three replicates) within each column with different letters are significantly different (P < 0.05), Duncan’s test. nd: not detected

399

The enthalpy required for melting of V-type crystal (∆HV-type) was also measured in non-stored control

400

sample within the range of 12.9 J/g. There is no significant difference between non-stored control and MI-

401

25 regarding ∆HV-type parameter (P > 0.05) (Table 3). However, the ∆HV-type was much higher for non-stored

402

MI-50, MI-75 and MI-100 with a value of 53.6, 56.4 and 50.9 J/g, respectively. This is ascribed to the

403

presence of more crystals of V-type with higher levels of modified inulin, which would result in an increase

404

in the enthalpy. Therefore, these samples need more energy to melt, resisting higher temperatures without

405

melting, giving to the chocolate more stability at the higher storage temperatures (Rodriguez Furlán et al.,

406

2017).

20

407

As can be clearly seen in Fig. 4, the melting peak of sucrose undergoes a major change. In the chocolates

408

formulated with modified inulin, a remarkable decrease was found in the mid-point temperature of the

409

sucrose peak. This could be linked to a faster crystallization induced by inulin addition, acting as a

410

nucleating agent in the chocolate matrix. On the other hand, the ∆H of sucrose peak in inulin-added

411

samples notably decreased. In this regard, ∆H of sucrose in non-stored control sample decreased from

412

122.8 j/g to 59.5 and 29.1 j/g about MI-50 and MI-75, respectively. This is basically due to less amounts of

413

sucrose with regard to replacement by modified Inulin. It is necessary to note that substitution of 100%

414

modified inulin led to the total disappearance of the endothermic peak of sucrose.

415

The thermograms of stored chocolates exhibit changes that take place in the proportions of polymorphic

416

forms during storage (Fig. 4). It is evident that V-type crystal was integrated to VI-type form in stored control

417

and MI-25 chocolates. In general, a certain phase difference was prominent in these samples, in which

418

form V had transformed to form VI, proposing a phase shift or separation in chocolate over three months of

419

storage. In contrast, V-type crystal form was still the most abundant polymorphism type in the stored MI-50

420

and MI-75, showing they preserved their polymorphs. From thermograms, stored chocolate with 100%

421

modified inulin was on the verge of transitioning to form VI according to thermal analysis, albeit with

422

existence of much more levels of V-type crystal.

423

The DSC thermograms also revealed that the onset temperature (To) of VI peaks for storage chocolate

424

formulated with dodecenyl succinylated inulin at the lowest level (25%) was increased compared to its non-

425

stored sample. Furthermore, melting point of VI peak in control and MI-25 showed the higher value with

426

respect to their non-stored ones. The increase in melting peak indicated that chocolate stored for three

427

months might be due to continued crystallization of more stable crystal form upon storage. In contrast,

428

there was no change in onset temperature of V and VI peaks of MI-50 and MI-75 with respect to their non-

429

stored samples. In the same way, stored MI-50 and MI-75 chocolates showed an identical melting peak as

21

430

their relevant non-stored samples (P > 0.05) (Table 3). It could be concluded that the melting behavior of

431

MI-50 and MI-75 did not considerably change during storage time.

432

It can be also observed that enthalpy value, as shown in Table 3, varied among the stored samples, which

433

was associated with the different energies required to complete the melting of fat crystals. The peak area of

434

the stored control sample considerably increased during storage, which might be due to the post

435

crystallization. Consequently, the shift to higher peak area was probably the result of the re-crystallization

436

of V-type to VI-type. Likewise, the enthalpy of stored MI-25 was increased compared to its non-stored

437

chocolate. On the other hand, it is clear that the substitution of modified inulin at higher levels (50, 75 and

438

100%) seemed to slow down this post crystallization process during storage. The peak area of VI-type

439

crystal for MI-50 and MI-75 even after three months was still quite low, indicating that V crystals were

440

abundant polymorphism type. Total replacement of sugar by modified inulin (MI-100) did not also produce a

441

statistically significant change in ∆H of V and VI forms with respect to non-stored MI-100 (P > 0.05).

442

Therefore, grafted alkyl groups on the inulin backbone could mostly interact with triglycerides in cocoa

443

butter and the more polar central polymeric chain end interacting mostly with the surface of sucrose,

444

causing thus the necessary free energy reduction that avoids destabilization and further phase separation

445

of the suspension upon storage (Do et al., 2010). Overall, these results propose that the effect of modified

446

inulin on the stabilization of chocolate is not due to change in rheological properties resulted by introducing

447

the biopolymer into chocolate matrix and is relatively owing to interaction of its functional groups with solid

448

particles, which is in accordance with previous reports about influence of biopolymeric surfactant on

449

chocolate functional properties (Ceballos et al., 2016).

450

3.5. Change in distribution of fat crystals

451

To further explain the change in the rheological, thermal and structural properties of chocolate variants,

452

XRD was used to characterize the crystalline network of control and low-calorie samples. It has been

453

established that phase transitions in chocolate are directly related to the presence of biopolymeric 22

454

surfactant with different concentration, as well the storage time (Ceballos et al., 2016). XRD diffractograms

455

of fat crystals in chocolates with different ratios of modified inulin are shown in Fig. 5. As visualized, non-

456

stored control sample displayed mixture of V- and VI-types crystalline structure with some characteristic

457

diffraction peaks located at 2θ = 21.2°, 2θ = 22°, 2θ = 23.4° and 2θ = 24°, whose d-spacing (d001)

458

determined at values of 5.1, 4.7, 4.1 and 3.9 Å, respectively. This is in agreement with Biswas, Cheow, Tan

459

and Siow (2017), who measured the d-spacing of dark chocolate in the range of 3.9-5 Å. In the inulin-added

460

samples, two significant peaks appeared on the XRD pattern of chocolate at the angles of 2θ = ~ 4.6°

461

(d001 = 14.8 Å) and 2θ = ~ 8.1° (d001 = 11.8 Å), which correspond to the characteristic diffraction peaks of

462

modified inulin (Kiumarsi et al., 2019). As the result of XRD analysis, non-stored MI-25 presented mixture of

463

V- and VI-type fat crystals, while MI-50, MI-75 and MI-100 showed mainly V-type. The diffraction pattern of

464

the latter samples also shows multiple pronounced peaks located at 2θ = 21.4° (d001 = 5.2 Å), 22.1°

465

(d001 = 4.8 Å) and 23.8° (d001 = 4.2 Å) and two characteristic peaks at 2θ = ~ 4.6° (d001 = 14.8 Å) and 2θ = ~

466

8° (d001 = 11.8 Å). Confirmation of the transition was also in line with DSC thermograms of chocolates.

467

The relative crystallinity of chocolate variants was also obtained from X-ray diffractograms (see

468

Supplementary Material). After replacing modified inulin at the level of 25%, the relative crystallinity of

469

chocolate from an initial value of 54.4% marginally increased to about 56.1%. This is due to the

470

appearance of new peaks at 2θ = ~ 4.5° and 2θ = ~ 8.1° (related to inulin characteristic peaks) and 2θ =

471

22.2° and 2θ = 23.4° (related to V-type crystalline pattern), and also 2θ = 22.2° and 2θ = 24.4° (associated

472

with VI-type crystalline pattern). A substantial increase in the relative crystallinity was determined for non-

473

stored MI-50, as its value increased to 58.1%. This might be attributed to, first, presence of new peaks after

474

inulin introducing and second, developing new linkages in the amorphous region of chocolate, which

475

causes an increase in the matrix crystallinity (Kiumarsi et al., 2019). However, relative crystallinity of non-

476

stored MI-75 and MI-100 was obtained to be closer to non-stored control sample (about 54.5%), which

477

could be explained by a loss in the pronounced peak of sugar. 23

478

479

Fig. 5. XRD patterns of non-stored (left) and stored chocolates (right) as the influenced by modified inulin substitution. Dotted

480

arrows indicate the position of fat crystals.

481 482

XRD patterns of stored chocolates as a function of modified inulin replacement are also presented in Fig. 5.

483

Control storage sample showed mainly VI-type crystalline pattern characterized by strong reflections at 2θ

484

= 23.4° and 2θ = 24°. Compared to non-stored control, the characteristic peak located at 2θ = 21.2° and 2θ

485

= 22° were completely disappeared, which was coincident with the conversion of V-type to VI-type

486

crystalline with losing the chocolate crystallinity. It is shown that the VI-type fat crystal, the typical blooming

487

form, which is created by the conversion of V-type, could represent bad characteristics to consumers

488

(James & Smith, 2009). Regarding stored MI-25, in the transition from V to VI, the first peak (2θ = 21.2°)

489

became less pronounced and the second (2θ = 22°) and third peaks (2θ = 23.4°) were integrated to form a

490

single peak in comparison with non-stored MI-25, signifying a rearrangement in crystal packing. In contrast,

491

diffractogram of stored MI-50 was found not to change, nor was seen an emergence of a new peak. This

492

sample showed three strong diffraction peak at 2θ = 21.4°, 2θ = 22.1° and 23.3º, indicating V-type

493

polymorphs and a minor peak at 2θ = 24.8º, corresponding to the characteristic of VI-type crystal. Likewise, 24

494

stored MI-75 was characterized with a predominance of the V form because the peaks at 2θ = 20.7° and

495

22° were more expressive (Fig. 5). Regarding this sample, the peaks at 2θ = 23.4° and 2θ = 24° were also

496

almost unchanged upon storage. Furthermore, with respect to non-stored MI-75, the first diffraction peak

497

was slightly shifted toward lower degree (from 2θ = 21.1° to 2θ = 20.8°) along with an increased peak

498

height tending to the V polymorphism. All these results indicate that a minor change in the polymorphic

499

transformation of chocolates formulated with 50% and 75% modified inulin upon storage. The process of

500

stabilization might be ascribed to the fact that hydrophobically modified inulin coats the solid particles

501

spreading into the lipid continuous phase producing a steric stabilization (Do et al., 2010; Aidoo et al.,

502

2013). Therefore, the higher stability produced by modified inulin might be owing to its effect as an actual

503

biopolymeric surfactant, giving the stabilization of particle phase dispersed in a fat-based suspension.

504

However, regarding stored MI-100, the peak intensity at 2θ = 23.4 ° and 2θ = 24.2° slightly increased,

505

indicating that some VI-type crystal was still present. These results were in accordance with the thermal

506

behavior obtained from DSC test, as described in Section 3.4.

507

As a consequence, introducing of modified inulin at the levels of 100% slowed the transition of V to VI form

508

upon storage, showing mainly V-type form with a little amount of VI-type form after three storage months.

509

The presence of 50% and 75% modified inulin nearly halted the transition of form V to VI upon storage.

510

However, modified inulin at the level of 25% could not prevent in the transition V-type crystal to VI-type

511

from. After three months of storage, the proportions of VI-type crystal were ∼72%, ∼18% and ∼21% for

512

chocolates with 25%, 50% and 100% modified inulin, respectively, whereas the proportion of form VI in

513

chocolate with 75% modified inulin was <15%.

514

3.6. Sensory properties

515

3.6.1. QDA evaluation

516

The mean intensities for the sensory attributes of chocolate as a result of modified inulin replacement and

517

storage time are presented in the spider diagrams in Fig. 6. Among the low-calorie chocolates produced in 25

518

this study, the samples formulated with intermediate levels of inulin (50% and 75%) showed the highest

519

intensities in the terms of gloss and brown color (P < 0.05) and no significant difference was observed after

520

storage time (P > 0.05). In general, intensities of brown color and gloss of stored MI-50 and MI-75 were the

521

most similar to their non-stored samples (P > 0.05). In contrast, control and MI-25 samples showed the

522

lowest intensities of gloss and brown color among non-stored chocolates (P < 0.05) and these attributes

523

were significantly declined after storage (P < 0.05).

524 525

Fig. 6. The mean intensities of QDA profiling evaluated by trained panelists.

526

As a general observation, the blooming area and blooming color of the stored chocolates with 50%

527

modified inulin or higher were similar to the corresponded non-stored samples. In contrast, the highest

528

intensities of blooming area and blooming color were found in control and MI-25 samples after storage

529

period. These results are important because blooming attribute is considered as the main appearance

530

sensory characteristic for chocolate, playing a crucial role in sensory acceptability of this product. As has

531

been commented on above (microscopy section), modified inulin at the levels of 50-100% could help to 26

532

prevent the blooming in the chocolate matrix and so, showing no development of visible surface crystals

533

after three months of storage. Based on previous studies (Shah et al., 2010), chocolates with induced

534

blooming tend to decrease in all sensory quality traits, especially appearance, for which the responses of

535

consumers changed most sensitively and radically. In the present study, cracking attribute on the surfaces

536

of chocolate variants was evaluated and the results showed that the crack regions were not observed in MI-

537

50, MI-75 and MI-100 even after storage, while the highest intensity of this defect was found in control and

538

MI-25 as a result of instability of solid particles, and also phase separation in chocolate suspension during

539

storage (Fig. 6).

540

In the QDA, the chocolates made with the higher levels (50-100%) of modified inulin were found to have a

541

decrease in sweetness intensity (sweet taste and sweet aftertaste), but the reduction in sweet taste was

542

similar in magnitude for both stored and non-stored samples (P > 0.05). This is mainly ascribed to less

543

amounts of sucrose with respect to substitution by modified inulin. It is worth noting that the sweetness of

544

the sample formulated with 25% inulin was not significantly different with non-stored control (P > 0.05).

545

Upon storage, the sweetness of control and MI-25 declined, while the bitterness was perceived more

546

intense in these samples. Compared to control sample, chocolate made with the highest level of inulin (MI-

547

100) showed a notable increase in bitterness and bitter aftertaste, which this change was smaller for non-

548

stored samples than storage samples (P < 0.05). The higher perception of these attributes is due to the

549

bitter taste of modified inulin used in the formulations (Shah et al., 2010; Kiumarsi et al., 2019). The natural

550

bitterness of modified inulin probably led to a lower perception of sweet taste and sweet aftertaste in the

551

chocolates containing this component, which might also have masked the sweet aftertaste in the samples

552

containing sucrose.

553

The textural sensory profile of chocolate as affected by modified inulin constitution and storage condition is

554

also illustrated in Fig. 6. Increasing inulin content with simultaneous reduction in sucrose resulted in an

555

increase in astringency of all inulin-added chocolates (P < 0.05). There was no significant difference 27

556

between stored and non-stored MI-50 and MI-75 regarding perceived astringency in the mouth (P > 0.05).

557

Compared to non-stored control and MI-25 samples, the chocolates with higher inulin contents (50-100%)

558

exhibited lower intensities (P < 0.05) of powderiness and mass forming and higher intensities (P < 0.05) of

559

smoothness. There was no significant difference among the stored and non-stored samples (P > 0.05).

560

After storage, powderiness and mass forming of control and MI-25 samples increased while the

561

smoothness declined (P < 0.05).

562

The differences in chewiness, snap and firmness attributes of stored chocolates are also evident in Fig. 6.

563

Chewiness and snap attributes were perceived stronger by increasing modified inulin ratio. Regarding

564

firmness attribute, non-stored MI-75 and MI-100 showed the stiffer texture than other inulin-added

565

chocolates. In the meantime, firmness, chewiness and snap of stored MI-50 and MI-75 were the most

566

similar to non-stored chocolates, while stored control and MI-25 samples exhibited the lowest intensities for

567

these attributes (Fig. 6). As mentioned before, modified inulin at the lowest content (25%) decreased the

568

crystallinity of storage chocolate (as shown by XRD and DSC) and contributed to formation of a less stable

569

system, which produced a poor structure. Moreover, by referring to previous points, the functional

570

properties of chocolate containing modified inulin at the higher ratios were almost unaffected upon storage,

571

in which these samples could preserve their polymorphism, resulting in a formation of a proper chocolate

572

matrix. Additionally, microstructural examination showed that chocolates with higher amounts of inulin (50-

573

100%) had a much denser network structure and high solid packing intensity accounting than that of lowest

574

content (see section 3.3). The Pearson’s correlation test indicated that sensory firmness was positively

575

related to instrumental elastic modulus parameters (r = 0.94), complex modulus (r = 0.89), enthalpy

576

(r = 0.95) and relative crystallinity degree (r = 0.95) (P < 0.05).

577

From Fig. 6, modified inulin increased the melting rate of non-stored chocolates (excluding MI-25) and

578

replacement of higher amounts of inulin caused further increase in this attribute. The storage chocolates

579

formulated with 50% and 75% inulin were more comparable to non-stored samples regarding melting rate 28

580

(P > 0.05). As discussed, biopolymer surfactants can effectively coat the surfaces of the sugar and cocoa

581

particles dispersed in chocolate matrix to maintain or enhance the flowability of molten chocolate, where it

582

took a shorter time for the chocolates with inulin at intermediate levels to melt (Do et al., 2010). However, in

583

the stored control and MI-25 chocolates, melting rate was significantly decreased compared to their non-

584

stored samples (P < 0.05). It should be noted that chocolate samples containing higher levels of inulin

585

showed a slight change in the polymorphic transformation of the crystalline domains during storage, so the

586

observed higher melting rate is likely to have contributed by anti-blooming effect of inulin. This was

587

paralleled by melting behavior results obtained by DSC determination. This information is important as it

588

provides information on oral melting behavior with an impact on release of flavor components and also oral

589

sensation during consumption of food products (Aidoo et al., 2013). The Pearson’s correlation test

590

indicated that sensory melting rate was positively related to instrumental elastic modulus parameters

591

(r = 0.94), complex modulus (r =0.89), melting point (r =0.95) and relative crystallinity degree (r = 0.95)

592

(P < 0.05).

593

Fig. 6 also shows the differences between the odor and flavor of chocolate variants perceived by the

594

panelists in QDA assessment. The results indicated that inulin substitution and storage time influenced the

595

sensory perception of odor and flavor in terms of cocoa and buttery. In the inulin-added samples, cocoa

596

and buttery (odor and flavor) were more perceptible in non-stored MI-50 and MI-75 samples, while the

597

lowest intensities were perceived in non-stored MI-25 sample. After storage condition, the odor and flavor

598

of stored samples with higher levels of modified inulin (50-100%) were remained at the levels of their non-

599

stored samples (P > 0.05), whereas stored control and MI-25 chocolates exhibited lower intensities of

600

cocoa and buttery (odor and flavor) than that of non-stored ones (P < 0.05).

601

3.6.2. Acceptance test

602

The consumers (n=120) evaluated the overall liking of the chocolate samples as influenced by modified

603

inulin substitution and storage condition. Overall acceptance of all non-stored chocolates formulated with 29

604

modified inulin was in the domain of excellent or very good (≥ 6) (Kiumarsi et al., 2019). According to the

605

obtained scores, the non-stored MI-50 (8.1) and MI-75 (8.4) presented the highest scores for the overall

606

acceptance, followed by non-stored control and MI-25 samples with a score of 7.5, whereas the sample

607

formulated with modified inulin at the level of 100% received the lowest acceptability (6.5) (P < 0.05). The

608

strong expressions of bitterness and astringency perceptions in the MI-100 declined the overall acceptance

609

of this sample. Moreover, the lack of sugar component in the formulation of MI-100 might be the main

610

reason for the lowest overall acceptance. Shah et al. (2010) observed that the addition of inulin results in

611

good acceptance of low-calorie chocolates. Golob et al. (2004) also verified that the substitution of sucrose

612

with inulin in chocolate formulations did not negatively influence the acceptance of this product. On the

613

other hand, the stored chocolates formulated with modified inulin at the levels of 50% (7.8) and 75% (8)

614

presented the best acceptance scores, while control (4.0) and MI-25 samples (4.7) were less acceptable

615

between the stored ones (P < 0.05). In this regard, stored MI-100 chocolate exhibited the acceptable score

616

with the intermediate value of 6.

617

3.6.3. Relationship between the descriptive attributes and the acceptance test

618

The relationship between sensory attributes (resulting from QDA) and overall liking (resulting from

619

acceptance test) was investigated by means of PLS modeling. Commonly, the overall acceptance was the

620

dependent variable (Y-matrix), and the QDA attributes were the independent variables (X-matrix)

621

(Tenenhaus, Vinzi, Chatelin, & Lauro, 2005). The purpose of PLS was to evaluate the positive and negative

622

attributes that were mainly related important to the consumers acceptance of the low-calorie chocolate

623

variants. When the standard deviation of a given attribute does not cross the-axis, it can be considered a

624

major positive or negative attribute at a 95% confidence level. The extent of the columns indicates the

625

importance of each attribute to the consumer, whether positive or negative.

626

The attributes positioned on the positive part of Y-axis represent the positive importance on the

627

characterization of the chocolate, while the columns of the negative part of the Y-axis interfere in a negative 30

628

way in the acceptance (Tenenhaus et al., 2005). From Fig. 7, the consumers’ attitude was affected by the

629

attributes gloss, brown color, blooming area, blooming color, smoothness, powderiness, mass forming,

630

cracking, sweetness, bitterness, sweet aftertaste, bitter aftertaste and cocoa odor. The attributes gloss and

631

brown color positively influenced the consumer acceptance, as also observed by (Cadena et al., 2013) in

632

chocolate, possibly because these descriptors are important characteristic in the appearance of chocolate.

633

The descriptors sweetness, bitterness and their related aftertastes influenced negatively the consumers’

634

attitude, owing to the purpose of this study that explained to them. The terms of low-calorie and presence of

635

natural sweetener might have motivated the consumers to give greater importance to these attributes,

636

which is frequently highlighted in low-calorie products. After analyzing the standard deviation and extend of

637

the columns, it was also found that blooming area, blooming color and cracking had pronounced negative

638

effect on consumers’ opinion (Fig. 7). Moreover, the consumers’ acceptance was positively affected by

639

smoothness of samples and also cocoa odor. On the other hand, perceiving powderiness and mass

640

forming in the mouth during oral processing negatively influenced the consumer assessment (Fig. 7).

31

641 642

Fig. 7. Partial least squares regression coefficients (black = attribute that contribute positively to consumer acceptance; white =

643

attribute that contribute negatively to consumer acceptance; gray = attribute without significant contribution to consumer

644

acceptance).

645

The results of external preference mapping are presented in Fig. 8. According to the results, the non-stored

646

inulin-added samples showed the higher consumers’ acceptance with regard to having the most desirable

647

texture, appearance, odor and flavor characteristics, except MI-100 that showed the lowest acceptance duo

648

to the highest bitterness, astringency and the lowest sweetness compared to other samples. With respect

649

to taste, texture, odor and flavor, non-stored samples differed from the other stored ones (P < 0.05), being

650

more acceptable in the consumer evaluation. However, the chocolates with intermediate levels of modified

651

inulin (MI-50 and MI-75) were more accepted due to having the highest cocoa flavor and odor without

652

presenting significant differences (P > 0.05) between samples before and after storage. Moreover, the

653

lowest powderiness and mass forming and the highest smoothness were perceived by panelists during the 32

654

consumption of MI-50 and MI-75. This demonstrated that for these samples, the mentioned attributes were

655

the ones that most influenced the consumer acceptance. This might be also related to anti-blooming effect

656

of modified inulin at these levels as explained previously. In contrast, both stored control and MI-25

657

samples were less acceptable due to declining gloss, sweet taste and increasing crack and blooming area

658

on their surfaces after storage time (P < 0.05). Moreover, the acceptability of these samples was negatively

659

affected by increasing the powderiness and mass forming attributes.

660 661

Fig. 8. External preference map determined by PLS regression from QDA evaluation and respondents’ overall liking scores for

662

the 21 sensory attributes of non-stored (a) and stored samples (b). Diamond symbol = chocolate samples and plus symbol =

663

consumers.

664 665

4. Conclusion

666

The present study displays the potential of producing low-calorie chocolates by using sucrose substitutes

667

with modified inulin as biopolymer surfactant and bulk sweetener. Chocolates formulated with higher ratios

668

of modified inulin presented higher plastic viscosity, flow behavior index and elastic modulus. Low-calorie

669

chocolates containing intermediate modified inulin contents exhibited same melting point and crystallinity

670

after three months of storage. The stored chocolates with intermediate levels of modified inulin did not 33

671

exhibit any change in thermal behavior. Polymorphisms of fat crystals in the stored chocolates with

672

intermediate ratios of modified inulin were also remained at the level of their non-stored samples. As a

673

result of QDA evaluation, sensory perception of stored chocolates having intermediate content of

674

dodecenyl succinylated inulin was considerably improved and remained the same intensities that their non-

675

stored samples. It could generally be observed that stored samples with intermediate hydrophobically inulin

676

contents were most accepted by the panel over all inulin-added chocolates regarding their sensory

677

attributes. According to our results, the presence and preservation of gloss, brown color, smoothness and

678

cocoa odor, and the lack or suppression of sweetness and bitterness are important for high acceptance of

679

low-calorie chocolate. Therefore, the present results can support the chocolate industry in meeting the

680

needs of a new, more demanding consumer niche, searching for low-calorie products with the desired

681

sensory attributes after long-term storage.

34

682

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37

Highlights: Modified inulin ratios affect rheological, thermal and sensory properties of chocolate. Modified inulin addition inhibited the blooming phenomenon in chocolate upon storage. Crystalline pattern of chocolates with intermediate levels of inulin was unchanged after storage. Instrumental findings can support but cannot replace sensory evaluations. Modified inulin is demonstrated to be a promising ingredient for chocolate industry.

Conflict of interest The authors declare no conflict of interest.