Characterization of β-carotene loaded emulsion gels containing denatured and native whey protein

Journal Pre-proof Characterization of β-carotene loaded emulsion gels containing denatured and native why protein Yao Lu, Like Mao, Hongxia Zheng, Hongqiang Chen, Yanxiang Gao PII:

S0268-005X(19)32160-5

DOI:

https://doi.org/10.1016/j.foodhyd.2019.105600

Reference:

FOOHYD 105600

To appear in:

Food Hydrocolloids

Received Date: 18 September 2019 Revised Date:

22 November 2019

Accepted Date: 16 December 2019

Please cite this article as: Lu, Y., Mao, L., Zheng, H., Chen, H., Gao, Y., Characterization of β-carotene loaded emulsion gels containing denatured and native why protein, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2019.105600. 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.

Graphical abstract

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Characterization of β-carotene loaded emulsion gels containing

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denatured and native why protein

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Yao Lu, Like Mao*, Hongxia Zheng, Hongqiang Chen, Yanxiang Gao

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

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Laboratory for Food Quality and Safety, College of Food Science & Nutritional

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Engineering, China Agricultural University, Beijing, China

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Corresponding author:

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Tel: +86-10-62737034

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Fax: +86-10-62737986

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E-mail: [email protected] (L. Mao)

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Abstract:

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The current study developed whey protein emulsion gels containing β-carotene, and the

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effects of the content of denatured protein and native protein were investigated. Two

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sets of emulsion gels were obtained by fixing the total protein content or the denature

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protein content (Cprotein-D). Emulsion properties were mostly affected by the content of

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native protein (Cprotein-N), and higher Cprotein-N resulted in emulsions with smaller

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particle size, higher surface charge and better creaming stability. Emulsions were then

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gelled with the addition of Glucono-δ-lactone. At fixed total protein content, higher

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Cprotein-D contributed to gels with higher mechanical properties, e.g., fracture stress,

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Young’s modulus and storage modulus (G′). At fixed Cprotein-D, the increase in Cprotein-N

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also resulted in stronger gels but at much lower magnitude. The dynamic gelation

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analysis revealed that the increase in Cprotein-N or Cprotein-D resulted in shorter gelling

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time. All the gels had rather high water holding capacity, and the gels with greater

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mechanical strength had lower swelling ratios. When emulsion gels were applied as

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carriers for β-carotene, both Cprotein-D and Cprotein-N had significant effects on the light

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stability and heat stability of β-carotene.

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Keywords: emulsion gel; denatured protein; native protein; gel structure; β-carotene

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1. Introduction

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Emulsion gels are structured emulsion systems, which have dispersed phases entrapped

50

within three-dimensional (3D) network (Farjami & Madadlou, 2019). Yoghurt, cheese,

51

and some processed meat are common food with emulsion gel structures. Emulsion

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gels are generally prepared through two-stage processes, i.e., emulsification and

53

gelation. The gelation can be originated from proteins, polysaccharides or their

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mixtures, via heat-induced, salt-induced, acid-induced, or enzyme-induced approaches

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(Lu, Mao, Hou, Miao, & Gao, 2019). When the gelling agents have surface activity

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(e.g., whey protein, soy protein, gum Arabic), they may also behave as emulsifiers to

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stabilize the emulsions. In other cases, emulsifiers and gelling agents can be different

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ingredients. Emulsion gels are usually applied to structure food systems, offering

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desirable rheological and textural properties, and they are also widely used in

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fat-reduced food (Dickinson, 2012). Many recent studies have revealed that emulsion

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gels are suitable carriers for lipid-soluble food bioactives, as the strong gel network can

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provide effective protection, and the modulation of gel structures allows controlled

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release and digestion of the incorporated ingredients (McClements, 2010; Torres,

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Murray, & Sarkar, 2016; Lu, et al., 2019).

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In emulsion gels, compositions and structures of the interface and continuous phase

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have great influence on the their functionality. According to the different interactions

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between the interfacial membrane and the continuous gel, the dispersed oil droplets can

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behave as active or inactive fillers (Dickinson, 2012; Lu, et al., 2019). With active

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fillers, the dispersed phase also participates the assembly of the gel network, and it is

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able to reinforce gel structure. Generally, emulsion gels using proteins to stabilize the

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interface and gel the continuous phase have active fillers. In these systems, the

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reduction in oil droplet size contributed to larger interfacial area, and thus led to gels

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with higher strength (Sala, van Vliet, Cohen Stuart, van de Velde, & van Aken, 2009).

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Literature studies also indicated that droplet coalescence or oil solidification could

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assist the formation of stronger gels (Oliver, Wieck,

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Scholten, & van Aken, 2015). Furthermore, the interface can largely influence droplet

77

distribution and emulsion stability, which may also affect the structures of the gels

78

(Guido, van Aken, Cohen Stuart, & van de Velde, 2010). Although different proteins

79

(e.g., whey protein, soy protein, sodium caseinate) have been tested for their roles in the

80

properties of emulsions gels, no detailed information regarding the effects of the

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content and structures of proteins at the interface were available (Lu, et al., 2019). With

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inactive fillers, the oil-water interface has minor or no interactions with the continuous

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gel body, and the oil phase is likely to weaken the gel structure. Generally, the gel

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systems containing low molecular weight surfactants have inactive fillers (Dickinson,

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2012).

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The biopolymers (i.e., proteins, polysaccharides) in the continuous phase constitute the

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main structures of the gel network, and their structures and content play essential roles.

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In a typical cold-gelation process, proteins are preheated to unfold their ternary

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structures and expose hydrophobic residues. The denatured protein molecules then

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aggregate to form 3D network with the aid of gelation triggers, e.g., acid, salt or

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enzymes (Mao, Lu, Cui, Miao, & Gao, 2019). Generally, emulsion systems with higher

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protein content are more favorable for the formation of compact gel network because of

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the presence of larger number of crosslinking sites. Second, with large number of oil

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droplets dispersed in protein dispersion, the protein concentration threshold for gelation

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can be well reduced, compared to pure gel systems. Mao et al. (2014) reported that a

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system with a protein content of 4% was able to form emulsion gels with

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self-supporting behaviors. Third, at a fixed protein concentration, protein preheating of

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& Scholten, 2016; Oliver,

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higher intensity (e.g., longer heating time, higher heating temperature) could facilitate

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the formation of denser gel network (Mao, Miao, Yuan, & Gao, 2018). In some

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emulsion gels, there could be two or more proteins and/or polysaccharides

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(Balakrishnan, Nguyen, Schmitt, Nicolai, & Chassenieux, 2017; Feng, Jia, Zhu, Liu, &

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Yin, 2019). In these systems, the interactions between biopolymers had critical impact

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on the development and structures of the gels. Depending on the charging properties,

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proteins and/or polysaccharides could form gel systems with single continuous phase,

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double continuous phase, uniform continuous phase, or non-homogeneous chained gel

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systems (van de Velde, de Hoog, Oosterveld, & Tromp, 2015). Furthermore,

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biopolymer-biopolymer interactions were likely to change during the gelation as

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affected by pH, ionic strength, etc., which then affected gel structures (Williams,

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Phillips, Matiamerino, & Dickinso, 2004). However, less attention was paid to the gel

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systems containing non-gelling biopolymers.

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In the current study, native whey protein was used mainly as an emulsifier, and

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heat-denatured whey protein was used as a gelling agent. β-Carotene loaded emulsion

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gels were prepared by varying the content of denatured protein and native protein, and

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both emulsion properties and gel properties were characterized, aiming to better

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understand the roles of oil-water interface and continuous phase in the structure and

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functionality of the gel systems. To the authors’ knowledge, it is the first time to study

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emulsion gel systems with one type of protein at both native and denatured states,

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which is meaningful for the innovation of healthy food.

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2. Materials and methods

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2.1. Materials

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Corn oil was the product of Changshouhua Food Company Limited (Shandong, China),

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and used withoug additional purification. Bipro-type whey protein isolate(WPI) was 5

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purchased from Davisco Foods (Le Sueur, MN, USA). Crystalline β-carotene with a

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purity >99% was a favor of NHU Holding Group Co., Ltd. (Zhejiang, China). Sodium

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azide, glucono-δ-lactone (GDL), n-hexane were products of Sigma-Aldrich (St. Louis,

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MO, USA).

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2.2. Preparation of denatured protein powder

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To induce protein denaturation, WPI dispersion (5 wt%) was incubated in a water bath

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at 85 °C for 30min, and then cooled rapidly to about 25 °C using ice. The samples were

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lyophilized using a vacuum freeze dryer (Marin Christ, Germany), and the obtained

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protein powder was stored in a desiccator for further use.

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2.3. Determination of interfacial tension

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Protein powder was hydrated in deionized water and kept overnight. Then interfacial

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tension between protein solution (1 wt%) and corn oil was measured using a

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Tensiometer K100 (Krüss, Hamburg, Germany) following a classical Wilhelmy plate

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method. During the measurement (20 °C), a platinum Wilhelmy plate (19.9×10×0.2

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mm) was immersed in the protein solution (20 mL) to a depth of 3 mm at a surface

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detection speed of 15 mm/min. The vessel drive speed applied for the tracking of the

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liquid surface was regarded as the surface detection speed. When the surface was

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determined by the microbalance in the machine, the vessel moved at the selected

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surface detection speed to the position specified by the immersion depth (3 mm).

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Afterwards, an interface between the protein solution and corn oil was created by

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carefully inject the corn oil over the protein solution (O'Sullivan, Rellano, Pichot, &

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Norton, 2014). Evolution of interfacial tension during the measurement of monitored,

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and the values at equilibrium were reported.

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2.4. Emulsion preparation

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The method for the preparation of emulsions was previously reported by Mao, Roos &

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Miao (2014). WPI (native) dispersions of different protein content were mixed with

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corn oil (20 wt%) using a high speed blender (Ultra-Turrax, IKA, Germany) to form

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coarse emulsions, which were then homogenized at 500 bar for three passes using a

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high pressure homogenizer (Niro-Soavi, Parma, Italy). The emulsions were rapidly

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cooled to about 25 °C using ice. The lyophilized denatured protein powder was added

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to the emulsions, and mixed well for complete hydration of the powder.

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For the preparation of β-carotene emulsions, β-carotene crystals (6 wt%) were first

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dissolved in the oil phase at 140 °C before mixing with the water phase. Samples

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containing different content of native protein (Cprotein-N) and denatured protein (Cprotein-D)

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were listed in Table 1.

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2.5. Characterization of WPI stabilized emulsions

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Droplet size and surface charge of the emulsions were determined by utilizing a

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Malvern Zetasizer (Nano-ZS90, Malvern Instrument, UK). RI (refractive index) of

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aqueous and oil phases were set to 1.33 and 1.45, respectively. In order to reduce

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multiple scattering effect, each sample was diluted with deionized water prior to the

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measurement. Droplet size was described as hydrodynamic diameter (nm) and

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polydispersity index (PDI), and surface charge was described as ζ-potential (mV).

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Viscosity of the emulsions was evaluated via a DHR-2 rheometer (TA Instruments,

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UK) , and the geometry with parallel plates (40 mm diameter and 1 mm gap) was

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selected. The determination was carried out at the shear rate range of 10–400 s−1 at

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25 °C.

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Emulsion stability was determined using a LUMiSizer (L.U.M.GmbH, Berlin,

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Germany), which applied centrifugation force to accelerate the test. The machine used

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near-infrared light (880 nm) to scan the whole samples, and intensity of the transmitted 7

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light was recorded. The measurement (25 °C) was set at a centrifugation speed of 4000

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rpm and the samples were scanned 800 times at an interval of 10 s. The curves of

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integrated intensity of the light transmitted as a function of time were plotted, and

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slopes of the curves were regarded as instability index (Mao, Roos, & Miao, 2015).

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2.6. Preparation of emulsion gels

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Emulsion gels were prepared following the method reported previously (Mao, et al.,

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2014). Briefly, GDL (0.5 wt%) was introduced into emulsions and mixed well with

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stirring bars, and the mixtures were incubated quiescently at 25 °C for 12 h to form the

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gels.

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2.7. Characterization of emulsion gels

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2.6.1. Viscoelastic behaviors

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The same rheometer for viscosity analysis was also applied to the evaluation of

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viscoelastic characteristics of the gels. Emulsion drops were placed onto the plate right

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after the addition of GDL, and the dynamic gelation process was evaluated at a strain of

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0.5% and a frequency of 1 Hz under the oscillation mode (25 °C). The linear

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viscoelastic range of the samples was pre-determined through a strain sweep. The

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evolution of storage modulus (G′) and loss modulus (G′′) in the gelation process were

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recorded (Mao et al., 2018).

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2.6.2. Textural properties

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Textural analysis was performed for all the emulsion gels using a FTC texture analyzer

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(Rockville, Maryland, USA). Samples for the test were specifically formed in plastic

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containers (34 mm id., 50 mm height) following the approach reported earlier. The gels

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received uniaxial compression by a 12 mm cylindrical plunger at a strain of 50%.

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Fracture stress, fracture strain and Young’s modulus of the samples were then

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calculated. All the reported values were means of eight replicates.

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2.6.3. Swelling ratio

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Swelling ratios of the gels were measured according to a study by Carvajal-Millan et al.

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(2015). The gels were cut into cylindrical shape and weighed, and the initial mass was

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recorded as M0. All the samples were immersed in deionized water for 24 h, and then

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the swollen gels were moved out for mass determination (M). The swelling ratio (t) was

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calculated using the following equation: t = (M-M0)/M0 * 100

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2.6.4. Water holding capacity (WHC)

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WHC of the gels was measured following the method reported by Wang and

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co-workers (2018) with slight adjustments. The emulsions were gelled in 10 mL

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centrifuge tube and weighed (W0). Afterwards, samples were centrifuged at 14,000 rpm

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and 25 °C for 20 min. The water released was removed, and the mass of the remaining

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gels (with centrifuge tube) was weighed and recorded as W. WHC (%) of the emulsion

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gels was calculated using the equation below: WHC=(W0-W)/W0 *100

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2.6.5. Scanning electron microscopy (SEM) observation.

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Microstructural observation of the samples was carried out using a SU-8100 SEM

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(Hitachi, Japan). The gel structures were fixed in a glutaraldehyde-cacodylate buffer,

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followed by ethanol dehydration and critical point drying. The dried samples were then

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placed on a copper stud and sputtered with gold for the observation.

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2.8. Stability of β-carotene

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2.8.1. UV-light stability

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Light stability test was performed in a Q-SUN xenon test chamber (Q-Panel Lab

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Products, USA) at an irradiance of 0.68 W/m2. Fresh emulsion gels were first moved

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into transparent glass bottles (flushed with nitrogen), and then incubated in the test

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chamber for 8h at 25 °C (Liu, Wang, Xu, Sun, & Gao, 2016). Sampling was done each

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2h to determine the remained content of β-carotene.

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2.8.2. Thermal stability

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Glass bottles (flushed with nitrogen) containing fresh emulsion gels were incubated at

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25 and 55 °C for 15 d at dark. Sampling was done every 3d to determine the remained

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content of β-carotene.

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2.8.3. Determination of the content of β-carotene

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Emulsion gels from stability test were mashed to destroy the gel structures for the ease

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of the extraction of β-carotene with n-hexane. β-Carotene content was then determined

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using a UV 1800 spectrophotometer (Shimadzu, Japan) by recording the absorbance at

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450 nm, in assistance with a standard curve (Yuan, Gao, Zhao, & Mao, 2008).

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Retention ratio C/C0 (C: content of β-carotene after storage; C0: initial content of

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β-carotene before storage) of β-carotene in the gels after the test was applied to describe

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the stability of β-carotene.

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By fitting a first-order kinetic model, the degradation kinetics of β-carotene was also

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evaluated. Reaction rate constant (k) and half-life (t1/2) of β-carotene was calculated

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following the equations below (Sharma, Kaur, Oberoi, & Sogi, 2008): C / C 0 = exp(−k • t )

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ln C = ln C 0 − kt

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t1 / 2 = ln 2 / k 242

2.9. Statistical analysis

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Mean values ± standard deviations (SD) were reported for all data points based on three

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replicates. An analysis of variance (ANOVA) of the data was performed using Origin

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Pro 8 at a level of significance of 5%.

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3. Results and discussion

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3.1. Characterization of the emulsions

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It is known that proteins in the continuous phase or at the interface can largely affect

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emulsion properties (McClements, 2004). However, there is little information available

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when a mixture of denatured protein and native protein is used. Table 2 summarizes the

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droplet characteristics of the emulsions, i.e., droplet size, PDI and surface charge, as

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affected by the content of proteins at the interface and continuous phase. At fixed total

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protein content (group A), the increase in Cprotein-N led to smaller particle size and lower

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PDI of the emulsions, as higher interfacial coverage prevented droplet association via

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steric hindrance and electrostatic repulsion (McClements, 2015). In fact, the magnitude

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of zeta-potential was also higher in the emulsions with higher Cprotein-N. At fixed

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Cprotein-D (group B), similar trends were also observed, and the increase in Cprotein-N

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contributed to emulsions with smaller particle size. At fixed Cprotein-N, the change in

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Cprotein-D did not affect particle size significantly (A1 and B1, A3 and B3). Therefore,

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the content of native protein played a more significant role in emulsion properties. The

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interfacial tension between protein solution and corn oil was also determined, and the

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result indicated that the native protein (13.748±0.029 mN/m) was capable to reduce the

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interfacial tension a lower level than the denatured protein (14.935±0.006 mN/m) at

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equilibrated state, which was in agreement with literature study (Euston, Finnigan, &

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Hirst, 2000). Besides, native protein was firstly added to form the emulsions, followed

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by the addition of denatured protein. Therefore, the native protein was more likely to

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dominate the interface. However, the emulsions with lower Cprotein-D had significantly

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higher magnitude of surface charge (B1>A1, A3>B3). There could be some

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interactions between denatured protein and native proteins (Vardhanabhuti, Foegeding,

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McGuffey, Daubert, & Swaisgood, 2001), and the resultant complex might have

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decreased amount of charged moieties. For example, the exposed thiol groups in

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denatured protein can interact with the surface active groups of native protein (Torres,

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Janhøj, Mikkelsen, & Ipsen, 2010). However, the underlying mechanisms required

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further investigation.

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Fig. 1 shows the difference in viscosity between the two groups of emulsions. The

276

results indicated that all the emulsions were shear-thinning fluids, and viscosity of the

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emulsions decreased gradually with the increase in shear rates (Dybowska, 2004).

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Shear thinning behavior was a sign of droplet flocculation in emulsions, which

279

presented resistance against shearing at lower force and broke up at high shear rates

280

(Floury, Desrumaux, & Lardières, 2000). Dickinson et al. (1997) believed that

281

reversible depletion flocculation caused by unabsorbed protein present in the

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continuous phase can cause pseudoplasticity of the emulsion. In the current systems,

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there was also a high content of denatured protein at the continuous phase, which was

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possible to induce droplet flocculation (Euston et al., 2000). Second, as protein

285

denaturation had thickening effect, the increase in Cprotein-D contributed to the systems

286

with higher viscosity (Fig. 1A) (Britten, Giroux, Jean, Rodrigue, 1994). At fixed

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Cprotein-D, the change in Cprotein-N did not affect emulsion viscosity significantly (Fig. 1B).

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It is known that emulsions with smaller particles generally have higher viscosity

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(McClements, 2010), which was not observed for the present samples, probably due to

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the bigger roles of Cprotein-D.

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All the emulsions had good creaming stability before gelation, probably due to the high

292

content of protein, and the higher interfacial charge. We performed acceleration tests to

293

observe the differences in emulsion stability by applying a multisample centrifugation

294

technique. Fig. 2 reveals that emulsions with higher Cprotein-N generally had lower

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instability index, both at fixed total protein content and Cprotein-D.

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3.2. Characterization of the emulsion gels

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3.2.1. Viscoelastic properties of the emulsion gels

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The gelation process was simulated in a rheometer, and the evolution of the rheological

299

properties of the systems was evaluated (Fig.3). In the current study, all the systems

300

followed similar gelation dynamics, which could be divided into three stages. At the 1st

301

stage, both G' and G'' of the systems were low and increased slowly. In this stage, the

302

hydrolysis of GDL was just initiated, and pH of the systems was still high, and there

303

was little interaction between protein aggregates; At the 2nd stage, both G' and G'' got

304

sharp increase, and G' was increased even faster. In this stage, pH of the systems was

305

lowered to the isoelectric point (pI) of the protein, and the protein molecules tended to

306

form 3D network as a result of hydrophobic attraction, van der Waals forces and

307

hydrogen bonding (Ye & Talor, 2009; Mellema, van Opheusden, & van Vliet, 2002). In

308

some studies, the time point at which the sharp increase started was refered to as gelling

309

time (Ersch et al., 2015); At the 3rd stage, the increase of G' and G'' was well slowed

310

down until levelled off. In this stage, pH of the systems was kept at low level (Mao et al.,

311

2014), and the development of the gel network gradually reached a steady state. At

312

fixed total protein content, the system with the highest Cprotein-D (A1) started all the three

313

stages much earlier than the other two systems, because of the earlier onset of gelation.

314

Furthermore, G' at the steady state was higher in the systems with higher Cprotein-D

315

(A1>A2>A3) (Fig. 3A). These results indicated the big role of denatured protein in the 13

316

development of the gel network. With higher Cprotein-D, there were more binding sites

317

for aggregate association, leading to faster gelation and higher gel strength. At fixed

318

Cprotein-D, the increase in Cprotein-N (with higher total protein content) resulted in faster

319

gelation and bigger G' at the steady state (Fig. 3B). The findings indicated that the

320

native protein took part in the development of the gel network, and higher total protein

321

content promoted gelation. It has been well documented that denatured protein at the

322

interface participate the gelation process with the denatured protein at the continuous

323

phase (Ye & Talor, 2009; Mao et al., 2018). However, there was little information on

324

the roles of native protein in the gelation of denatured protein. In fact, the current

325

systems could be regarded as mixed emulsion gels with two proteins. The native

326

protein in the current systems was not possible to gel individually, because of its folded

327

structures and low concentration. On the other hand, no phase separation was observed

328

when the two proteins coexisted in the dispersions, indicting the two proteins were

329

thermodynamically compatible. Therefore, there were other mechanisms responsible

330

for the participation of the native protein in the gel network: 1) Native protein (either in

331

the continuous phase or at the interface) formed complexes with denatured protein

332

(through electrostatic or hydrogen bonding) (Britten, Giroux, Jean, & Rodrigue, 1994;

333

Zhai, Day, Aguilar, & Wooster, 2015); 2) Native protein behaved as fillers to support

334

the gel network. With higher density of protein deposited at the interface, the oil

335

droplets could have higher density and strengthen the gel structures (Lu, et al., 2019);

336

the native protein could compete for water binding and concentrated the denatured

337

protein (Ersch, ter Laak, van der Linden, Venema, & Martin, 2015). Wu et al. (2018)

338

concluded that non-networking proteins (mainly soy protein peptides) did not affect the

339

G′ of the emulsion gels (heat-induced), because the non-networking proteins were

340

dissolved and located at the pores of gel network. However, in the current study, native

14

341

protein was believed to be mostly distributed at the interface. Second, different gelation

342

methods and protein structures in the two studies could also be responsible for the

343

contradicting results. It was deducted that the oil droplets worked as active fillers in

344

these systems (Dickinson, 2012). However, systems with smaller oil droplets (e.g., A3)

345

did not always had higher G', probably because the size effect was blocked by the big

346

role of protein content.

347

3.2.2. Textural properties

348

Gel food received compression and stretching during oral processing, and the forces

349

received by the brain were largely perceived as texture (Stokes, Boehm, & Baier, 2013).

350

Table 3 represents the textural characteristics of the emulsion gels. It was observed that

351

the content of denatured protein and native protein greatly affected the texture of the

352

gels. At fixed total protein content, the increase in Cprotein-D was accompanied by the rise

353

in fracture stress and Young’s modulus (A1>A2>A3), indicating the big role of the

354

denatured protein in the formation of gel structures of high strength. Similar results

355

were also obtained by Vardhanabhuti and coworkers (2001), who found that the

356

replacement of native whey protein by denatured protein resulted in gels with higher

357

fracture stress and modulus. It was reported that Young’s modulus could reflect

358

molecular rigidity, which for biopolymers, could be a sign of the number of cross-links.

359

With higher Cprotein-D, there were more cross-linking sites in the systems, leading to

360

higher rigidity of the gels (Saavedra Isusi, Karbstein, & van der Schaaf, 2019). At fixed

361

Cprotein-D, only slight increase in Young’s modulus and fracture stress (B3>B2>B1) with

362

the increase in Cprotein-N was observed. In the current study, gelation was originated

363

from the aggregation of protein molecules, which was favorable when the molecules

364

were unfolded (at denatured state). For example, by changing the Cprotein-D from 4% (B1)

365

to 4.5% (A1), the fracture modulus was increased by 18.06% (from 2.99 to 3.53 MPa);

15

366

by changing the Cprotein-N from 1% (B2) to 1.5% (B3), the fracture stress was only

367

increased by 1.31% (from 3.06 to 3.10 MPa). These results also indicated that the

368

current systems were homogeneous, as inhomogeneity usually resulted in a decrease in

369

fracture stress, as result of stress concentration (van Vliet & Walstra, 1995).

370

Interestingly, fracture strain of the gels was independent on Cprotein-D and Cprotein-N,

371

probably because of the soft nature of the gels at the current protein range.

372

3.2.3. Microstructures

373

Figure 4 illustrates the morphology of the emulsion gels through SEM observation,

374

which clearly showed the differences in the microstructures of the samples. At fixed

375

total protein content (group A), the gels with higher Cprotein-D had more compact

376

structures with smaller pores. In sample A1, the gel network was formed by protein

377

aggregates of smaller size and similar shape. In sample A3, the aggregates were rather

378

big with different shapes. The results reconfirmed the bigger roles of denatured protein

379

for the development of compact gel network. At fixed Cprotein-D, the changes in Cprotein-N

380

only resulted in slight changes in the microstructures of the gels. Comparatively, the

381

gels with the highest Cprotein-N had the densest structure and lowest number of pores.

382

Whey protein could develop particulate gels or filamentous gels, which was mainly

383

dependent on the force balance between attraction and repulsion among protein

384

molecules (Lefevr & Subirade, 2000; Ikeda & Li-Chan, 2004). In the current study,

385

lower repulsion force was present as the pH of the gels was lowered to the pI of the

386

protein, and the development of particulate gels was favored. Oil droplets could not be

387

clearly observed in these SEM images, because they were completely surrounded by

388

the particulate protein network. In fact, there were a lot of small-sized protrusion at the

389

surface of the gel network, which could be the oil droplets (Lu et al., 2019).

390

3.2.4. Swelling ratio & WHC

16

391

The three-dimensional network in emulsion gels entrap a certain amount of water, and

392

they are also able to adsorb addition water (Begam, Nagpal, & Singhal, 2003), which

393

are important for the mouth feel of gel food and the release of functional ingredients

394

incorporated (Lu et al., 2019). Swelling properties of gels were highly dependent on the

395

pore size and water-adsorbing capacity of the gelling biopolymers (Feng et al., 2019).

396

Fig. 5A reveals that the gels with higher Cprotein-D (fixed total protein content) had lower

397

swelling ratios. For example, the swelling ratio of A1 was 1.64%, and that of A3 was

398

4.46%. As discussed earlier, higher Cprotein-D contributed to more compact gel network,

399

and there could be only small pores to accommodate additional water. Second,

400

denatured protein had decreased capability to adsorb water (Saffon, Britten, & Pouliot,

401

2011). Third ， the gels already had high amount of water adsorbed during the

402

development of gel network. In fact, all the three gels maintained their self-supporting

403

structures after the swelling test. Fig. 5B indicates that gels with lower Cprotein-N (fixed

404

Cprotein-D) had higher swelling ratios. The greater adsorption of water might be resulted

405

from the bigger size of the oil droplets (Feng et al., 2019). Morphological observation

406

showed that the gels with higher swelling ratio became softer and were not able to

407

maintain their intact structures. During food digesting, swelling of gels can facilitate

408

bioactive release (Mao et al., 2019).

409

Water holding capacity (WHC) refers to the ability of the tested samples to immobilize

410

moisture through capillary forces, which is largely dependent on pore size (Wu et al.,

411

2009). Fig. 6 indicates all the samples had quite big WHC (>85%), and there was small

412

difference among samples. At fixed total protein content, the highest WHC (87.29%)

413

was observed in the sample with the highest Cprotein-D. At fixed Cprotein-D, the highest

414

WHC (88.78%) was found in the sample with the highest Cprotein-N. Some studies

415

concluded that WHC and storage modulus of emulsion gels had positive association

17

416

(Sok Line, Remondetto, & Subirade, 2005; Yang, Liu, & Tang, 2013), which was also

417

found in the current study. Emulsion gels with higher storage modulus generally had

418

denser and more uniform microstructures, which allowed the gels to hold water

419

molecules strongly against centrifugation (Hu et al., 2013). In the current systems,

420

water was mostly bound to proteins, and only small amount of water could be present in

421

the pores of the gel network. Vardhanabhuti et al. (2001) suggested there could be a

422

transition in network structures from particulate to a more filamentous structures in gels

423

with more denatured protein, and the latter type of gels had higher ability to hold water.

424

3.3. Emulsion gels as carriers for β-carotene

425

β-carotene has strong antioxidant activity, and it is able to enhance the specific and

426

non-specific immune function. However, β-carotene is extremely sensitive to light,

427

heat and oxygen, and is prone to degradation, which can result in the loss of its

428

bioactivity (Rodriguez-Amaya, 2015). It has been proved that delivery systems,

429

including emulsions, gels, nanoparticles are able to protect β-carotene from

430

degradation (Mao, Wang, Liu, & Gao, 2018).

431

Fig. 7 shows the changes in the retention ratios of β-carotene within emulsion gels of

432

different protein content during the light stability test. At fixed total protein content

433

(Fig. 7A), the emulsion gels with higher Cprotein-D can retain more β-carotene

434

(A1>A2>A3). For example, in the gel with a Cprotein-D of 4.5%, about 56.53±0.03% of

435

the original content of β-carotene was retained after the test. However, only 45.06 ±

436

0.02% β-carotene was retained in the sample with a Cprotein-D of 3.5%. As discussed

437

earlier, gels with higher Cprotein-D had denser structures, which could effectively prevent

438

the contact of the embedded functional ingredients with external environmental stresses.

439

Second, carotenoids can form complexes with protein through hydrophobic

440

interactions (Dufour, &Haertlé, 1991;Wackerbarth, Stoll, Gebken, Pelters, Bindrich, 18

441

2009), and the complexes could be more resistant to oxidation. At fixed Cprotein-D, the

442

stability of β-carotene was improved with the increase in Cprotein-N (B3>B2>B1). This

443

may be due to the fact that β-carotene degradation was initially started at the emulsion

444

interface (Liu et al., 2016). The increase in Cprotein-N can enhance the thickness of the

445

interfacial film and effectively inhibit the initiation and propagation of the oxidation

446

process (Waraho, McClements, & Decker, 2011). Second, there were some

447

antioxidative moieties in protein molecules, e.g., cysteyl residues, disulphide bonds and

448

thiol groups, which also worked to inhibit β-carotene oxidation (Mao et al., 2018).

449

When the emulsion gels were stored at different temperatures (25 °C and 55 °C), the

450

changes in the retention ratios of β-carotene followed similar trends as those observed

451

in the light stability test (Fig. 8). For example, after a 12-day storage test, the retention

452

rate of β-carotene in A1 was 57.61±0.01% (25 °C) and 45.61±5.55% (55 °C), and that

453

of A3 was 52.81±0.02% (25 °C) and 37.81. ±1.92% (55 °C). In general, gels with

454

higher gel strength and denser structures could provide stronger protection for

455

β-carotene against adverse stresses. Similar results were also observed in other studies

456

on different lipophilic bioactives (Lu et al., 2019). Although the increase in the content

457

of native protein and denatured protein were both helpful in retaining higher content of

458

the bioactive, higher Cprotein-D was more effective.

459

In order to get a systematic understanding of β-carotene degradation in these gel

460

systems, degradation kinetics was calculated (Table 4). It was found that β-carotene in

461

the gels containing higher Cprotein-D had smaller k and bigger t1/2 values during light

462

stability and thermal stability test. For example, when the emulsion gel had a Cprotein-D

463

of 4.5% and a Cprotein-N of 0.5% (A1), β-carotene had a k value of 77.4×10-4 h-1, and a t1/2

464

value of 8.95 h during the light stability test; the k value was 2.56×10-4h-1, and the t1/2

465

value was 270.70 h when the gels was stored at 55 °C. The emulsion gels with higher

19

466

Cprotein-D had better protection effect on the embedded ingredient, and the degradation

467

rates were slower. It was also found that the increase in Cprotein-N could reduce the k

468

value of β-carotene in the emulsion gels and enhance its protective effect. The

469

calculation clearly indicated that the increase in Cprotein-D was more effective in slowing

470

the degradation of β-carotene than the increase in Cprotein-N, suggesting that the gel

471

structures provided more protection for β-carotene than the interface.

472

4. Conclusion

473

The present work studied the influence of the content of native protein and denatured

474

protein in the structure and functionality of emulsion gels. With strong self-assembling

475

properties of the denatured protein molecules at pH close to its isoelectric point, the

476

changes in Cprotein-D greatly affected mechanical, water adsorption and holding

477

capacities. Consequently, the gels with higher Cprotein-D retained more β-carotene during

478

stability tests. The native protein also took part in the development of the gel network

479

and the protection for β-carotene, though it played relatively weaker roles. The study

480

revealed the strong influences of the gel structures on the functionality of the emulsion

481

gels, and appropriate structural designs in the gel matrix and at the interface could bring

482

in flexible delivery of bioactives incorporated in the systems. The knowledge obtained

483

in this study would provide novel information for the development of delivery systems

484

for functional food.

485

486

Acknowledgments

487

This research was funded by National Natural Science Foundation of China (No.

488

31701648).

489

20

490

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24

Table 1. Composition of emulsions/emulsion gels with different content of native protein and denatured protein.

Group

A

B

Sample codes

Cprotein-D

Cprotein-N

(w/w)

(w/w)

A1

4.5%

0.5%

A2

4%

1%

A3

3.5%

1.5%

B1

4%

0.5%

B2

4%

1%

B3

4%

1.5%

*A2 and B2 were the same sample. For the ease of understanding, different codes were used.

25

Table 2. Droplet characteristics of emulsions with different content of native protein and

denatured protein. Samples

Size

PDI

Zeta potential (mV)

A1

393.75±7.71

a

0.24±0.05

-43.32±2.56a

A2/B2

326.88±4.23b

0.23±0.04

-51.02±1.88b

A3

276.33±7.19c

0.19±0.07

-56.62±1.30c

B1

400.20±8.51a

-46.90±0.90d

B3

283.47±6.22c

0.24±0.07 0.18±0.01

-46.23±0.21d

Values with different lowercase letters in the same column indicate a significant difference among the values of p < 0.05. A2 and B2 were the same sample. For the ease of understanding, different codes were used.

26

Table 3. Texture properties of emulsion gels with different content of native protein and denatured protein.

Samples

Fracture stress (MPa)

Fracture strain

a

0.52±0.04

Young's modulus (MPa)

a

9.00±0.41a

A1

3.53±0.17

A2/B2

3.06±0.14b

0.47±0.04ab

8.83±0.20a

A3

2.99±0.07c

0.45±0.02b

8.11±0.31b

B1

2.99±0.06c

0.45±0.02b

8.52±0.25c

B3

3.10±0.22b

0.48±0.04ab

9.00±0.41a

Values with different lowercase letters in the same column indicated a significant difference (p < 0.05). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

27

Table 4. Degradation rate constant (k), half-life time (t1/2), regression coefficient of first-order kinetic model (R2) for β-carotene in emulsion gels at different storage conditions. R2

t1/2 (h)

k (10-4h-1) Samples UV-light

25 °C

55 °C

UV-light

25 °C

55 °C

UV-light

25 °C

55 °C

A1

77.41±0.29a

1.85±0.36a

2.56±0.35a

8.95

374.59

270.70

0.8198

0.8865

0.9307

A2/B2

91.83±0.23b

1.85±0.13a

2.71±0.05a

7.55

374.59

255.72

0.9183

0.9793

0.9987

A3

105.60±0.34c

2.24±0.11b

3.21±0.20b

6.56

309.38

215.89

0.8644

0.9913

0.9844

B1

115.82±0.33d

2.05±0.22b

3.23±0.15b

5.98

338.05

214.55

0.8911

0.9547

0.9909

B3

73.21±0.25e

1.47±0.16c

2.64±0.22a

9.47

471.43

262.5

0.8526

0.9511

0.9729

Values with different lowercase letters in the same column indicated a significant difference (p < 0.05). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

28

Figure 1

A

40 A1

Viscosity (mPa.s)

35

A2

30

A3

25 20 15 10 5 0 0

100

200 Shear rate (s-1)

300

B 40

B1

35 Viscosity (mPa.s)

400

B2

30

B3 25 20 15 10 5 0 0

100

200 Shear rate (s-1)

300

400

Fig. 1. Viscosity of the emulsions with different content of native protein and denatured protein (A: fixed total protein content; B: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

29

Figure 2

A

0.50

Instability index

0.40

0.30

0.20

0.10

0.00

B

A1

A2

A3

B1

B2

B3

0.50

Instability index

0.40

0.30

0.20

0.10

0.00

Fig. 2. Instability index of emulsions with different content of native protein and denatured protein (A: fixed total protein content; B: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

30

Figure 3

A 100000

G', G'' (Pa)

10000

1000

100 A1,G' A1,G'' A2, G' A2, G'' A3, G' A3, G''

10

1 0

2000

4000 Time (s)

6000

8000

B 100000

G', G'' (Pa)

10000

1000

100 B1,G' B1,G'' B2, G'

10

B2, G'' B3, G' B3, G''

1 0

2000

4000 Time (s)

6000

8000

Fig. 3. Gelation dynamics of the emulsion gels with different content of native protein and denatured protein (A: fixed total protein content; B: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

31

Figure 4 B1

A1

A2/B2

A3

B3

Fig.4. Microstructures of emulsion gels with different content of native protein and denatured protein. A2 and B2 were the same sample. For the ease of understanding, different codes were used.

32

Figure 5

A

6

Swelling ratio (%)

5 4 3 2 1 0 A1

A2

A3

B1

B2

B3

B6

Swelling ratio (%)

5 4 3 2 1 0

C A1

A2

A3

B1

B2

B3

Fig.5. Swelling properties of emulsion gels with different content of native protein and denatured protein. (A: fixed total protein content; B: fixed denatured protein content; C morphology of the samples after swelling test). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

33

Figure 6

A 100

a

b

c

A1

A2

A3

c

b

B1

B2

80

WHC (%)

60

40

20

0

B 100

a

80

WHC (%)

60

40

20

0 B3

Fig.6. Water holding capacity (WHC) of emulsion gels with different content of native protein and denatured protein (A: fixed total protein content; B: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

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Figure 7

A

B

Fig. 7. The retention ratios of β-carotene in emulsions gels with different content of native protein and denatured protein when exposed to light (A: fixed total protein content; B: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

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Figure 8

A

B

C

D

Fig. 8. The retention ratios of β-carotene in emulsions gels with different content of native protein and denatured protein when stored at 25 and 55 °C (A&B: fixed total protein content; C&D: fixed denatured protein content). A2 and B2 were the same sample. For the ease of understanding, different codes were used.

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Emulsion gels were prepared with both denatured protein and native protein. Emulsion properties were mostly affected by the content of native protein. The denatured protein played a leading role in the gelation process and structures of the emulsion gels. Both proteins were responsible for the stability of β-carotene.

We declare that we have no conflict of interest.

Author statement

The work presented in this paper has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.