Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties

Accepted Manuscript Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties Esmeralda Santillán-Urquiza, Miguel Ángel Méndez-Rojas, Jorge Fernando VélezRuiz PII:

S0023-6438(17)30170-6

DOI:

10.1016/j.lwt.2017.03.025

Reference:

YFSTL 6098

To appear in:

LWT - Food Science and Technology

Received Date: 30 November 2016 Revised Date:

11 March 2017

Accepted Date: 13 March 2017

Please cite this article as: Santillán-Urquiza, E., Méndez-Rojas, M.E., Vélez-Ruiz, J.F., Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.03.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the

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physicochemical and rheological properties.

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Esmeralda Santillán-Urquizaa*

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Miguel Ángel Méndez-Rojasb

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Jorge Fernando Vélez-Ruiza*

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Departamento de Ingeniería Química y Alimentos, Universidad de las Américas Puebla.

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San Andrés Cholula, Puebla 72820, México.

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San Andrés Cholula, Puebla 72820, México.

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Departamento de Ciencias Químico-Biológicas, Universidad de las Américas Puebla.

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[email protected]

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*Corresponding author: [email protected], [email protected]

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Abstract

24 Yogurt is a highly consumed dairy product, regarded as healthy. The objective of this study

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was to fortify a set-type yogurt with two levels of iron oxide, zinc oxide, and calcium

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phosphate nanoparticles. Minerals were also used to make a comparison between nano and

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micro-sized minerals, to determine their effect on the physicochemical and rheological

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properties during 28 days of storage. The pH decreased while acidity increased in all

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samples during storage. Density and moisture did not show differences between samples, or

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during storage. Color parameters showed variations in iron-fortified samples, whereas an

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increase in net color change through storage was recorded for all samples. Syneresis

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increased significantly in micro-mineral samples, being lower in nano-fortified ones; during

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storage the separation significantly increased in all samples. The Herschel-Bulkley flow

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model fitted well the non-Newtonian behavior of the yogurt. The yogurts fortified with

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calcium and zinc nanoparticles increased their consistency and firmness concerning to the

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other samples, both parameters decreased during storage in all samples; yield stress and

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flow index did not significantly change during storage. In vitro digestion analysis of the

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yogurt with nanoparticles showed more solubility than micro-minerals, for the three

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minerals. In general, nanoparticles showed advantages over conventional fortification.

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Keywords: yogurt, fortification, nanoparticles, rheological properties, physicochemical

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properties.

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Chemical compounds studied in this article

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Calcium phosphate (PubChem CID: 24441); iron oxide (PubChem CID: 14833); zinc oxide

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(PubChem CID: 14806).

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1. Introduction

50 Low intake or absorption of minerals like calcium, iron and zinc might generate

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deficiencies which in turn are related to many human health problems including stunted

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growth in children, weak bones, and immune system disorders. Food fortification could

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play a key role to overcome this problem. Yogurt has gained wide acceptance among

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consumers as it is perceived as a healthy product rich in nutrients such as calcium and high-

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quality proteins (Mckinley, 2005). However, as it is common with all dairy products, the

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content of iron and zinc is naturally very low (Mehar-Afroz, Swaminathan, Karthikeyan,

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Pervez, & Umesh, 2012). Due to its nature and widespread consumption, yogurt might be a

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suitable vehicle for these minerals.

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Several studies about the fortification of yogurt with minerals have been published

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in recent years (Gahruie, Eskandari, Mesbahi, & Hanifpour, 2015; Gupta, Chawla, Arora,

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Tomar, & Singh, 2015; Karam, Gaiani, Hosri, Burgain, & Scher, 2013; Ocak & Köse,

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2010). It is well known that fortification of yogurt with minerals as iron and zinc ions can

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chemically interact with various food ingredients. Induced chemical reactions can cause

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changes in physicochemical properties important for the quality of yogurt, such as syneresis

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as well as on rheological features; also off-flavors have been associated with fortification of

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dairy products. The quality of fortified dairy products depends on the selected mineral

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source, concentration and potential effects on physicochemical and functional properties on

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the food chosen as a carrier (Fayed, 2015). Thus, it is paramount to find alternatives to

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reduce the potentially undesirable effects of mineral fortification in dairy products while

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maximizing absorption and quality (Mehar-Afroz et al., 2012; Sharifi, Golestan, &

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Sharifzadeh, 2013). The use of nanomaterials in food fortification has experienced

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significant growth in recent years and is very promising (Santillán-Urquiza, Ruiz-Espinosa,

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Angulo-Molina, Velez-Ruiz, & Méndez-Rojas, 2017). This trend has been driven by the

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ability of these structures to improve bioavailability and solubility of active ingredients due

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to their large surface-to-volume ratio. That could be achieved without compromising other

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food properties (Sanguansri & Augustin, 2006).

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The core-shell nanostructures used for the fortification of the yogurt have been

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previously designed, prepared and evaluated by our research group. Santillán-Urquiza et

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al., (2015) reported inulin-coated nanoparticles with an inorganic iron and/or zinc oxide

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core. Inulin made these minerals more soluble and bioavailable while reducing their

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reactivity, avoiding thus possible detrimental effects (Dickinson, 2012), but these

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nanoparticles have been not tested yet in an appropriate carrier. Therefore, the goal of this

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study was to determine the effect of adding inulin coated calcium phosphate, iron oxide and

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zinc oxide nanoparticles in a set-type yogurt, evaluating potential changes in

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physicochemical and rheological properties right after manufacturing and during

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refrigerated storage, comparing the results with those of added with micro-sized minerals

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and a control yogurt.

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2. Materials and methods

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2.1 Materials

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Nanoparticles of CaHPO4, α−Fe2O3 and ZnO coated with inulin (Fructagave SP750

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Monterrey, México) and micro-minerals commercially available of CaHPO4, α-Fe2O3 and

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ZnO (Sigma-Aldrich, México) were used for fortification. Pasteurized whole milk (Alpura®,

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México) and skim milk powder (Svelty®, México) were used for preparation of the yogurt,

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as well as lyophilized microorganism Choozit® (Danisco, Mexico) containing:

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Lactobacillus delbrueckii spp. bulgaricus y Streptococcus salivarius spp. thermophilus. For

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the analysis of digestion, the enzyme pepsin (Golden Bell, México) and pancreatin (Sigma-

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Aldrich, México were also utilized.

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2.2 Methods

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2.2.1 Synthesis of inorganic nanoparticles

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Inorganic nanoparticles of zinc oxide (ZnO), hematite with zinc oxide (α−Fe2O3@ZnO)

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and CaHPO4, all coated with the polysaccharide inulin, were prepared accordingly to

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previously reported methods by Santillán-Urquiza et al. (2015).

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2.2.2 Characterization of nanoparticles

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The nanoparticles were characterized by powder X-Ray diffraction (XRD), Fourier

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transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and

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thermogravimetric analysis (TGA) (Yue- Jian, 2010; Santillán-Urquiza et al, 2015).

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2.2.3 Determination of solubility of nano and micro minerals by in vitro digestion

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The in vitro digestion protocol was applied to samples of Ca30N, Ca30M, FZ50N, FZ50M, Zn50N

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and Zn50M as described previously by Cilla, Perales, Lagarda, Reyes-Barbera, & Farre.

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(2008), with minor modifications and comprising two sequential steps: gastric and

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intestinal. To evaluate the gastric digestibility of nanoparticles and micro-minerals, the

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dissolution process of 8 g of the samples of fortified yogurt in a solution of HCl (6 mol /L)

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adjusted to pH 2 was followed. A solution of the enzyme pepsin (20 mg per gram of

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sample) was then added and the mixture incubated at 37ºC with stirring (120 agitations per

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min) for 2 h. At the end of this time, the mixture was kept on ice for 15 min to stop the

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enzyme digestion. For the intestinal digestion phase, the pH was raised to 6.5 with a

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solution of sodium bicarbonate (1 mol/L), and then, 5 mg per gram of sample of pancreatin

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were added; incubation continued for 2 h after the pH was adjusted to 7.2 with a solution of

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NaOH (0.5 mol/L). The samples were centrifuged at 1252 g for 20 min and filtered. The

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concentration of Zn (II) and Fe (III) ions was determined by atomic absorption

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spectrophotometry (Varian SpectrAA 220Fs, Midland, ON, Canada). The concentrations of

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the specified ions were measured in an air/acetylene flame for Zn and Fe and

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NO2/acetylene flame for Ca. The amount of metal ions released was calculated from a

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calibration curve previously obtained (Argyri, Birba, Miller, Komaitis, & Kapsokefalou,

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2009).

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2.2.4 Preparation of yogurt samples

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For the yogurt preparation, milk was standardized adding 6 g of milk powder per 100 mL

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of pasteurized milk. Then, a heat treatment was applied by raising the temperature of the

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milk at 90°C for 20 min and then cooling down to 40-45°C. After cooling at 42°C, the milk

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was added with the lyophilized culture directly and stirred for 10 min, and poured into 100

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mL plastic containers, being the same procedure for all samples; then the minerals (both,

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nanoparticles and micro-minerals) were added at a concentration as described in Table 1

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and stirred for 20 min (120 agitations/min) until complete dissolution. Subsequently, the

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milk with minerals was incubated at 45°C for 5 h until a pH of 4.6 was reached, as well as

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the control (Lee & Lucey, 2010), all yogurt samples were stored for 28 days at 4±1°C.

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2.2.5 Physicochemical analysis

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The pH was measured by a digital potentiometer (Beckman, Denver, CO, USA), previously

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calibrated, at room temperature. Moisture content was determined through water

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evaporation (method 16.032, A.O.A.C., 2000). Acidity was quantified by titration of 9 mL

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of sample using phenolphthalein and NaOH (0.1 mol equi/L) (method 16.023, A.O.A.C.,

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2000). Density was determined by a gravimetric method using Grease pycnometers

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(Fisherbrand, ON, Canada). The color of yogurt was measured in a Color Gard System⁄05

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color meter (Hunter Labs, Reston, VA, USA), previously calibrated with black and white

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plates, having standardized reflectance values of L = 93.82, a* = -3.58 and b* = 6.50 for the

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white plate, and expressed by the L, a, b Hunter parameters. The tests were conducted with

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samples of 20 mL each, and calculating the net color change by the next equation (Díaz-

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Jiménez, Sosa-Morales, & Vélez-Ruiz, 2004):

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∆E = [(L-L0)2+ (a*-a0)2+(b*-b0)2]0.5

(1)

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Where: L, a*, and b* are the measured parameters corresponding to the yogurt sample at a

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particular time and L0, a0 and b0 are the Hunter parameters for the control.

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All measurements were carried out by triplicate. Syneresis of the yogurt was determined through a centrifugation procedure.

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Approximately 10 g of yogurt was transferred into a 50 mL glass tube and centrifuged at

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176 g for 20 min at 10°C (Rojas-Castro, Chacón-Villalobos, & Pineda-Castro, 2007). The

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syneresis was estimated as the released whey over the original weight (Eq. 2) and was an

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average of three determinations.

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Syneresis = (weight of supernatant / weight of yogurt) *100

(2)

2.2.6 Rheological measurements

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Flow response of yogurt samples was carried out in a Brookfield viscometer (DV-III

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Brookfield Engineering Laboratories Inc., Middleboro, MA, USA). Shear stresses (τ) were

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determined at the correspondent shear rates (γ) obtained with 5, 10, 20, 30, 50, 60, 70, 80,

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90 and 100 rpm at 20ºC. The experimental data were fitted to a Herschel-Bulkley Model

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(Eq. 3) and Power Law Model (Eq. 4).

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τ = τ0 + K · γn

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τ = K · γn

(4)

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The three parameters, yield stress (τ0), flow behavior index (n) and consistency coefficient

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(K) of these mathematical models were used to characterize the flow behavior of yogurt

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samples (Ramírez-Sucre & Vélez-Ruiz, 2013). The root-mean-square error (RMSE) (Eq. 5)

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was used to determine which one of the two models was the best fitting.

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RMSE= [1/d Σdi=1 (τexp − τpred) 2] 1/2

(5)

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In this equation, τexp and τpred represent shear stress obtained experimentally and predicted

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by the two rheological models, being d is the number of experimental data.

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2.2.7 Texture analysis

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A texture profile analysis (TPA) to determine two parameters, hardness and cohesiveness,

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was determined using a Texture Analyzer TA.XT2 texture meter (Stable Micro Systems,

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Haslemere, Eng) using the software Texture Expert (v.1.22, 1999). Measuring the double

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compression force (N) in all samples of yogurt (50 mL, mm height) using a cylindrical

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body of 4.3 cm in diameter, descending at a speed of 0.5 mm/s, and reaching a depth of 20

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mm. All measurements were carried out at a temperature of 20ºC after 0, 7, 14, 21 and 28

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days of storage, also by triplicate (Díaz-Jiménez et al., 2004; Walia, Mishra, & Pradyuman,

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2009).

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2.2.8 Sensory analysis

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In order to determine the sensorial acceptance of fortified yogurts with nano and micro-

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minerals, a sensorial evaluation of seven yogurt samples was carried out with a panel of 30

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non-trained individuals, in two sessions, evaluating three samples in the first session and

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four samples in the second session. Sensory evaluation was based on a simple hedonic scale

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of nine points, 1 = dislike extremely to 9 = like extremely (Singh & Muthukumarapan,

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2008; Dello-Staffolo, Bertola, Martino, & Bevilacqua, 2004).

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Panelists evaluated the yogurt samples on the seventh day (to allow stabilization of

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the yogurt and any residual fermentation through refrigeration storage), in two sessions and

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based on five attributes: color, odor, taste, texture and overall acceptability. Using seven

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samples, natural or control yogurt and six fortified yogurts, three with nanoparticles (Ca30N, 10

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FZ50N, and Zn50N) and three with micro-minerals (Ca30M, FZ50M, and Zn50M). Three and four

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samples were given to the panelists in the first and second session, respectively.

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201 2.2.9 Experimental design

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Fortified yogurt was made with three minerals: calcium, iron, and zinc of two different

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sources: micro-minerals and nano-minerals (nanoparticles). Including 240 mg of calcium,

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7.50 mg of iron and 7.50 mg of zinc for micro-minerals. Whereas for nano-minerals 240

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and 120 mg of calcium, 7.50 and 3.75 mg of iron, and 7.50 and 3.75 mg of zinc in 100 mL

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of yogurt were incorporated into the 9 formulations (Table 1), in addition to the control. All

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samples of control and fortified yogurt were analyzed after preparation, and at 7, 14, 21 and

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28 days of storage.

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The response variables identified as physicochemical, rheological and textural

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properties were statistically examined with the Minitab software (v.16, Minitab Inc.,

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Pennsylvania, USA). Statistical analysis was performed using analysis of variance

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(ANOVA). And Tukey test was applied for multiple comparisons of the mean values.

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3. Results and discussion

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3.1 Characterization of nanoparticles

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Nanoparticles with an inorganic core have an average size in the range from 50 to 80 nm,

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being soluble in water. Inulin was selected as coating material due to its biocompatibility,

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biodegradability and bioactivity as a well-known prebiotic compound. The complete

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characterization of the nanoparticles has been previously reported (Santillán-Urquiza et al.,

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2015).

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3.2 Physicochemical determinations fortified yogurts

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3.2.1 pH and acidity

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In foods, the acidity indicates the content of free acids and other chemical compounds, due

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to this, in an acidic food as the yogurt; a decrement of pH indicates the release of lactic acid

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(hydronium ions H3O+) in the medium by lactic acid bacteria (LAB).

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Results for determinations of pH and acidity (Table 2) showed no significant

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differences between fresh formulations with nanoparticles and micro minerals and no

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differences were observed respect to the control. In the overall analysis for pH and acidity

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of all samples, no significant differences were found because the fortified yogurts have the

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same behavior as the control yogurt.

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The pH values showed a trend to decrease (P <0.05) during the storage period in all

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samples with values of 4.65 to 4.30 for samples at 28 days of storage (Table 2). The

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decrease in pH during the storage was due to the production of lactic acid by the bacteria

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present in the yogurt. The same behavior wherein the pH decrease during storage was

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reported by El-Kholy, Osman, Gouda, & Ghareeb. (2011), in an iron fortified yogurt and

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buffalo milk for ten days of storage. Acidity values increased significantly trough 28 days of storage in all samples,

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being in the range of 0.86 to 0.90 g/100 mL (Table 2). The values obtained are consistent

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but lower with those observed by Sanz, Salvador, Jiménez, & Fiszman. (2008); Drago &

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Valencia, (2002), they reported values of acidity higher than 1.0 for a yogurt enriched with

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asparagus fiber, and 0.80 to 1.80 g/100 mL of lactic acid in dairy products fortified with

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iron and zinc, respectively.

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3.2.2 Syneresis

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Syneresis is the separation of the phases in a suspension or mixture. It is a natural

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phenomenon that occurs in dairy products such as yogurt; it is an important attribute in

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determining the quality of yogurt and other dairy products (Ocak & Köse, 2010).

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Yogurts fortified with micro minerals (Ca30M, FZ50M, and Zn50M) had the highest

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values of syneresis with 46.52, 55.39 and 51.18 g/100 mL respectively, compared with

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yogurts fortified with nanoparticles and the control yogurt (Table 2). The samples fortified

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with nanoparticles were more stable; this can be attributed to the size of the nanoparticles

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and the presence of inulin, which promotes water retention due to its gel-like structure. This

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observation is in agreement with results obtained for yogurt fortified with iron, zinc, and

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magnesium as reported by Achanta, Aryana, & Boeneke. (2007).

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Syneresis showed significant differences (P <0.05) during storage (Table 2),

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increasing significantly in all samples. This effect may be related to a decreasing of pH

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below 4.6, that contributes to caseins rearrangement and water release (Lee & Lucey, 2010).

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These results are comparable with those reported by Diaz-Jimenez et al. (2004) who

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prepared a yogurt with fiber reporting values of syneresis from 45 to 65 g/100mL.

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3.2.3 Moisture

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Moisture determination showed no significant difference (P <0.05), as expected, among

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fortified yogurts with micro-mineral, nanoparticles and the control (Table 2). Moisture is

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not affected by the addition of minerals, indicating that the amount added is not high

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enough to cause variations in this parameter. During storage, moisture values ranged

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between 83 and 84 g/100 g (Table 2). That are similar to those reported by Karam et al.

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(2013), with moisture values in the range from 80 to 85% for a yogurt fortified with

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different milk protein powders at various concentrations.

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3.2.4 Density

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Density results did not show a significant difference (P <0.05) for all samples (Table 2).

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And as expected, density showed no changes during the storage for any sample (Table 3).

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Because density is related to moisture, and the total solids content in each sample was

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preserved. The results are comparable to those reported by Donkor, Henriksson, Vasiljevic,

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& Shah. (2007) and Singh & Muthukumarappan (2008).

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3.2.5 Color

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The comparative statistical analysis of the three color parameters based on the Hunter scale,

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for all samples are included in Table 3. For the L, a* and b* parameters, found in the fresh

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samples fortified with calcium and zinc and the control, no significant differences (P <0.05)

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were observed because the added minerals are of white color. On the other hand, the

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samples fortified with iron showed significant differences (P <0.05) compared to the

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control or to samples fortified with calcium and zinc. Because the iron oxide added to the

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formulation is red, it produced a decrease in luminosity (L), a significant increase in the

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parameter a* which indicates a tendency to red, and a reduction in the parameter b*

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showing a decrease in yellow color of the yogurt samples. This is agreement with Ramirez-

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Sucre & Vélez-Ruiz, (2013) that reported an increase in the parameter a* with values of -

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2.97 for the control and 4.26 for samples with high concentration of caramel and fiber in

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the yogurt formulation.

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During storage, a significant decrease (P <0.05) in the L and b* parameters was

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observed, while the parameter a* showed a significant increase (P <0.05) in all samples

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(Table 3). The presence of minerals during storage promotes the oxidation of lipids from

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the yogurt, thus decreasing the luminosity and causing changes in parameters a* and b*.

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These values are comparable to those reported by Achanta et al. (2007) in yogurts fortified

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with various minerals. The analysis of the change in a global parameter (∆E) through the

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storage, showed significant changes (P <0.05) in all samples (Table 3), as a result of the

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variations observed in the three color parameters for all samples.

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3.2.6 Rheological properties

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The flowing nature of yogurt systems may be appreciated in their rheograms (Figure 1).

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They show a characteristic flow behavior, indicating that the decreasing viscosity is not

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constant. The rheograms show that all samples including control were of plastic nature

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because the shear stress values did not start at zero. Ramírez-Sucre & Vélez-Ruiz, (2013)

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and Damin, Alcántara, Nunes, & Oliveira. (2009) among others, also have reported this

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non-Newtonian behavior in yogurt added with different ingredients. According to the

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RMSE values, the model with best fit was the Herschel-Bulkley model, showing the lowest

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values of error compared to the Power-Law model (Table 4). The results of the flow index

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are from 0.20 to 0.30 for the Power Law model and 0.22 to 0.33 for the Herschel-Bulkley

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model, showing no significant differences (P <0.05) for any of the samples fortified with

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micro-mineral, and nanoparticles, concerning the control (Table 5).

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All systems showed a plastic-shear thinning behavior; with flow index values lower

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than one. These values of n were comparable with those reported by Aportela-Palacios,

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Sosa-Morales, & Vélez-Ruíz. (2005) in which n ranged from 0.35 to 0.45 for fortified

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yogurt with calcium.

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The values of the consistency coefficient obtained with the Herschel-Bulkley model

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(KHB) at day 0 ranged from 5.01 to 7.55 Pasn. The samples fortified with nanoparticles

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Ca30N and Zn50N had a significant difference (P <0.05) and showed the highest KHB values

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when compared to the other concentrations and with the control (Table 5). This indicates a

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greater interaction, which may be associated with the presence and size of nanoparticles. It

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may have considered that sample Ca30N, favors the interaction of nanoparticles of calcium

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phosphate with the casein of yogurt and the presence of inulin promotes the consistency

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increasing (Sfakianakis & Tzia, 2014; Heaney, Rafferty, Dowell, & Bierman, 2005).

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In the case of fortified yogurt with zinc the significant increase in consistency can

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be associated with the interaction of zinc by binding to casein micelles, and more

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specifically to colloidal calcium phosphate; which enhances the consistency of the yoghurt

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in the presence of zinc (Drago & Valencia, 2002). These interactions did not occur in the

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presence of iron; it did not exhibit a significant influence (Table 5). The results of the consistency coefficient for the studied yogurt samples showed a

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significant decrease (P <0.05) during the storage (Table 5). This reduction trend in the

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coefficient of consistency of yogurt is primarily due to structural changes in the gel, in

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agreement with the loss of firmness or stiffness of the protein matrix (Lee & Lucey, 2010).

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This KHB trend trough storage has also been reported by Diaz-Jimenez et al. (2004) and

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Basak & Ramaswamy. (1994) for a low-fat yogurt with added fiber and yogurt enriched

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with pectin and fruit concentrates, respectively.

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Finally, during the storage period (Table 5), the results of flow index and yield

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stress did not differ significantly for all samples (P <0.05). Thus, the flow index and yield

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stress were not affected by the addition of minerals and neither by the presence of inulin in

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the analyzed formulations. The results are comparable to those reported by Peng, Serra,

337

Horne, & Lucey. (2009).

338

3.2.7 Textural analysis

339

Textural characteristics and rheological properties of coagulated dairy products are affected

340

by their structural components. According to Walia et al. (2009), the structural arrangement

341

of the network determines the textural characteristics of yogurt products, that is influenced

342

by factors such as composition and manufacturing processes.

AC C

EP

TE D

333

343

The results of firmness and cohesiveness are summarized in Table 6; in which

344

firmness was the necessary force to attain a given deformation, it is a commonly evaluated

345

parameter for yogurt texture. The firmness of yogurt increases in samples of higher

17

ACCEPTED MANUSCRIPT

concentration of mineral and principally in yogurts fortified with nanoparticles reflecting a

347

stronger gel structure. The firmest samples corresponded to Ca30N and Zn50N with values of

348

0.93 and 0.72 N respectively; these results may be attributed to the interaction of these

349

minerals with the protein matrix of the yogurt, becoming stronger than the other

350

formulations or the control yogurt. These obtained values were comparable to those of

351

firmness from 0.65 to 0.97 N for fortified yogurt with calcium reported by Damin et al.

352

(2009).

SC

RI PT

346

In semi-solid like yogurt, the cohesiveness represents how well the product

354

withstands a second deformation with respect to how it behaved under the first deformation.

355

The value of cohesiveness was higher for almost all fortified samples, Ca30N was -0.65 whit

356

respect to the – 0.42 of the control, and attributed to the added of nanoparticles in higher

357

concentration.

TE D

M AN U

353

The both textural parameters of fortified yogurt were obtained during storage after 0,

359

7, 14, 21 and 28 days (Table 6). In all samples a significant decrease in firmness and

360

cohesiveness parameters was observed, this reduction was also associated with changes in

361

the consistency coefficient during storage.

362

3.3 Solubility and sensory evaluation

363

The results of digestibility showed that the nanoparticles of calcium, iron, and zinc were

364

more soluble than those containing micro-minerals.

AC C

EP

358

365

On the other hand, the sensory test results suggest that fortified samples with

366

nanoparticles, showed significant differences compared to the fortified yogurts with micro-

367

minerals in all evaluated attributes. In which an important observation is: the levels of

18

ACCEPTED MANUSCRIPT

calcium and zinc can be incorporated into the yogurt samples without causing significant

369

changes in consumer acceptance. Singh & Muthukumarappan (2008), observed similar

370

results in their study of calcium-fortified yogurt, being the properties of fortified yogurt

371

similar to control.

373

The results and more details of digestibility and sensory analysis are available in the Supplementary Material.

SC

372

RI PT

368

374 4. Conclusions

376

This work led to the preparation of a yogurt fortified with nanoparticles as a potential

377

enriched food for consumption by humans that may contribute with Ca (II), Fe (III) and Zn

378

(II) ions. To determine the effect and differences between yogurt fortification with

379

nanoparticles and micro-minerals regarding a natural, an analysis of the physicochemical

380

and rheological properties were carried out on fresh and stored samples.

TE D

M AN U

375

In the results of digestibility the nanoparticles of calcium, iron, and zinc showed

382

more solubility than those containing micro-minerals. At the beginning the pH, acidity,

383

moisture and density were not significantly different, while the results of color parameters

384

showed significant differences only for fortified yogurt with iron. The syneresis showed a

385

significant decrease in some of the samples fortified with nanoparticles, which is

386

considered as an added value since the size and coating of nanoparticles favored water

387

retention. All the fortified yogurt and the control showed non-Newtonian behavior; the

388

Herschel-Bulkley model fitted better the flow response than the Power Law model and

389

without significant changes in the parameters n and τ0. The yogurts fortified with

AC C

EP

381

19

ACCEPTED MANUSCRIPT

nanoparticles of calcium and zinc showed significant differences in parameters such as the

391

consistency coefficient and also corresponded to the highest values of firmness for samples

392

containing iron and the control.

RI PT

390

Most of the tests except for density and moisture, showed changes during storage in

394

all samples because yogurt is a short shelf-life fresh product. In general, the samples

395

fortified with nanoparticles had advantages over conventional fortification using micro-

396

minerals and compared to the control as they present improvements in aspects that are

397

important to determine the quality of yogurt. In general terms, the fortified yogurt samples

398

were sensory well accepted, it was observed that the best scores were obtained by the

399

samples fortified with nanoparticles of Ca30N and Zn50N for all the attributes.

M AN U

SC

393

400 Acknowledgments

402

The author Santillán-Urquiza acknowledges financial support for her Ph.D studies in Food

403

Science from National Council for Science and Technology (CONACyT-México) and

404

Universidad de las Américas Puebla (UDLAP). We are thankful to Fernando Arteaga

405

Cardona (UDLAP) for his valuable participation and suggestions.

406 407 408

5. References

409

Achanta, K., Aryana, K.J., & Boeneke, C.A. (2007). Fat-free plain set yogurts fortified with

410

411

AC C

EP

TE D

401

various minerals. LWT- Food Science and Technology, 40, 424-429.

A.O.A.C. (2000). Official methods of analysis of AOAC. Gaithersburg, Maryland. E.U.A.

20

ACCEPTED MANUSCRIPT

Aportela-Palacios, A., Sosa-Morales, M.E., & Vélez-Ruíz, J.F. (2005). Rheological and

413

physicochemical behavior of fortified yogur, with fiber and calcium. Journal of Texture

414

Studies, 36, 333-349.

RI PT

412

Argyri, K., Birba, A., Miller, D., Komaitis, M., & Kapsokefalou, M. (2009). Predicting

416

relative concentrations of bioavailable iron in foods using in vitro digestion: New

417

developments. Food Chemistry, 113, 602–607.

SC

415

Basak, S., & Ramaswamy, H. (1994). Simultaneous evaluation of shear rate and time

419

dependency of stirred yogurt rheology as influenced by added pectin and strawberry

420

concentrate. Journal of Food Engineering, 21, 385-393.

M AN U

418

Cilla, A., Perales, S., Lagarda, M.J, Reyes-Barbera, E., & Farre, R. (2008). Iron

422

bioavailability in fortified fruit beverages using ferritin synthesis by Caco-2 cells.

423

Journal of Agricultural and Food Chemistry, 56, 8699–8703.

TE D

421

Damin, M.R., Alcántara, M.R., Nunes, A.P. & Oliveira, M.N. (2009). Effects of milk

425

supplementation with skim milk powder, whey protein concentrate and sodium caseinate

426

on acidification kinetics, rheological properties and structure of nonfat stirred yogurt.

427

LWT - Food Science and Technology, 42, 1744-1750.

AC C

EP

424

428

Dello-Staffolo, M., Bertola, N., Martino, M., & Bevilacqua, A. (2004). Influence of dietary

429

fibre addition on sensory and rheological properties of yoghurt. International Dairy

430

Journal, 14, 263–268.

21

ACCEPTED MANUSCRIPT

Díaz-Jiménez, B., Sosa-Morales, M.E., & Vélez-Ruíz, J.F. (2004). Effect of fiber adding y

432

fat decreasing on physicochemical properties of yogurt. Revista Mexicana de Ingenieria

433

Quimica, 3, 287-305.

434 435

RI PT

431

Dickinson, E. (2012). Use of nanoparticles and microparticles in the formation and stabilization of food emulsions. Trends in Food Science and Technology, 24, 4-12.

Donkor, O.N., Henriksson, A., Vasiljevic, T., & Shah, N.P. (2007). Rheological properties

437

and sensory characteristics of set-type soy yogurt. Agricultural and Food Chemistry, 55,

438

9868-9876.

M AN U

SC

436

Drago, S., & Valencia, M.E. (2002). Effect of fermentation on iron, zinc, and calcium

440

availability from iron-fortified dairy products. Journal of Food Science, 67, 3130-3134.

441

El-Kholy, A.M., Osman, M., Gouda, A., & Ghareeb, W.A. (2011). Fortification of yoghurt

442

TE D

439

with iron. World Journal of Dairy and Food Sciences, 6, 159-165.

Fayed, A. (2015). Health benefits of some physiologically active ingredients and their

444

suitability as yoghurt fortifiers. Journal of Food Science and Technology, 52 (5), 2512-

445

2521.

AC C

EP

443

446

Gahruie, H.H., Eskandari, M.H., Mesbahi, G., & Hanifpour, M. A. (2015). Scientific and

447

technical aspects of yogurt fortification: A review. Food Science and Human Wellness,

448

4, 1-8.

449

Gupta, C., Chawla, P., Arora, S., Tomar, S.K., & Singh, A.K. (2015). Iron

450

microencapsulation with blend of gum arabic, maltodextrin and modified starch using

22

ACCEPTED MANUSCRIPT

451

modified solvent evaporation method – Milk fortification. Food Hydrocollids, 43, 622-

452

628.

Heaney, R.P., Rafferty, K., Dowell, M.S., & Bierman, J. (2005). Calcium fortification

454

systems differ in bioavailability. Journal of the American Dietetic Association, 105, 807-

455

809.

RI PT

453

Karam, M., Gaiani, C., Hosri, C., Burgain, J., & Scher, J. (2013). Effect of dairy powders

457

fortification on yogurt textural and sensorial properties: a review. Journal of Dairy

458

Research, 80, 400–409.

461 462

M AN U

460

Lee, W.J., & Lucey, J.A. (2010). Formation and physical properties of yogurt. Asian Australasian Journal of Animal Sciences, 23, 1127-1136.

Mckinley, M. C. (2005). The nutrition and health benefits of yoghurt.

International

TE D

459

SC

456

Journal of Dairy Technology, 58 (1), 1-12.

Mehar-Afroz, Q., Swaminathan, K., Karthikeyan, P., Pervez, K., & Umesh, M. (2012).

464

Application of nanotechnology in food and dairy processing: An overview. Pakistan

465

Journal of Food Sciences, 22 (1), 23-31.

AC C

EP

463

466

Ocak, E., & Köse, S. (2010). The effects of fortifying milk with Cu, Fe and Zn minerals on

467

the production and texture of yoghurt. Journal of Food Agriculture and Environment, 8,

468

122-125.

23

ACCEPTED MANUSCRIPT

Peng, Y., Serra, M., Horne, D.S., & Lucey, J.A. (2009). Effect of fortification with various

470

types of milk proteins on the rheological properties and permeability of nonfat set

471

yogurt. Journal of Food Science, 74, C666-673.

RI PT

469

Ramírez-Sucre, M.O., & Vélez-Ruiz, J.F. (2013). Physicochemical, rheological and

473

stability characterization of a caramel flavored yogurt. LWT - Food Science and

474

Technology, 51, 233-241.

SC

472

Rojas-Castro, W.N., Chacón-Villalobos, A., & Pineda-Castro, M.L. (2007). Characteristics

476

of liquid strawberry yogurt from different relationships of cow and goat milks.

477

Agronomía Mesoamericana, 18, 221-237.

479

Sanguansri, P., & Augustin, M.A. (2006). Nanoscale materials development a food industry perspective. Trends in Food Science and Technology, 17, 547-556.

TE D

478

M AN U

475

Santillán-Urquiza, E., Arteaga-Cardona, F., Hernández-Herman, E., Pacheco-García P.F.,

481

González-Rodríguez, R., Coffer, J.L., Mendoza-Álvarez, M.E., Vélez-Ruiz, J.F., &

482

Méndez-Rojas, M.A. (2015). Inulin as a novel biocompatible coating: Evaluation of

483

surface affinities toward CaHPO4, α-Fe2O3, ZnO, CaHPO4@ZnO and,α-Fe2O3@ZnO

484

nanoparticles. Journal of Colloid and Interface Science, 460, 339–348.

AC C

EP

480

485

Santillán-Urquiza, E., Ruiz-Espinosa, H., Angulo-Molina, A., Velez-Ruiz, J.F., Méndez-

486

Rojas, M.A. (2017). Applications of nanomaterials in functional fortified dairy products:

487

benefits and implications for human health. A. M. Grumezescu (Ed.), in

488

Nanotechnology in the Agri-Food Industry, Nutrient Delivery (pp. 293-328).

489

Netherlands, Elsevier Inc.

24

ACCEPTED MANUSCRIPT

Sanz, T., Salvador, A., Jiménez, A., & Fiszman, S.M. (2008). Yogurt enrichment with

491

functional asparagus fiber. Effect of fiber extraction method on rheological properties,

492

colour, and sensory acceptance. European Food Research and Technology, 227, 1515-

493

1521.

RI PT

490

Sfakianakis, P., & Tzia, C. (2014). Conventional and innovative processing of milk for

495

yogurt manufacture; development of texture and flavor: a review. Review Foods, 3, 176-

496

192.

SC

494

Sharifi, A., Golestan, L., & Sharifzadeh, M. (2013). Studying the enrichment of ice cream

498

with alginate nanoparticles including Fe and Zn salts. Journal of Nanoparticles, 1, 1-5.

499

Singh, G., & Muthukumarappan, K. (2008). Influence of calcium fortification on sensory,

500

physical and rheological characteristics of fruit yogurt. LWT - Food Science and

501

Technology, 41, 1145-1152.

TE D

M AN U

497

Walia, A., Mishra, N., & Pradyuman, K. (2009). Effect of fermentation on

503

physicochemical, textural properties and yoghurt bacteria in mango soy fortified

504

yoghurt. African Journal of Food Science, 7, 120-127.

AC C

EP

502

505

Yue-Jian, C. (2010). Synthesis, self-assembly and characterization of PEG-coated iron

506

oxide nanoparticles as potential MRI contrast agent. Drug Development and Industrial

507

Pharmacy, 36, 1235-1244.

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ACCEPTED MANUSCRIPT Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties. Tables

Samples Minerals

RI PT

Table 1. Yogurts formulations, control and fortified 100 mL per portion.

84.02

Total solids (g/100g) 15.98

82.08

17.98

15

82.24

17.76

15

82.67

17.33

50/80

15

83.50

16.50

7.5/12

50/80

15

83.76

16.24

nano

3.7/6

25/40

15

82.70

17.30

nano

7.50

50

15

82.59

17.41

Size

Amount (mg)

RDI (%)

Inulin (mg)

0

0

0

0

0

Ca30N

Ca

nano

240

30

15

Ca30M

Ca

micro

240

30

Ca15N

Ca

nano

120

15

FZ50N

Fe/Zn

nano

7.5/12

FZ50M

Fe/Zn

micro

FZ25N

Fe/Zn

Zn50N

Zn

Zn50M

Zn

Zn25N

Zn

TE D

M AN U

SC

Control

Moisture (g/100g)

micro

7.50

50

15

82.96

17.04

nano

3.75

25

15

82.11

17.89

AC C

EP

Ca=calcium, Fe=iron, Zn=zinc, N=nanoparticles, M=micro-minerals *RDI (recommended daily intake).

1

ACCEPTED MANUSCRIPT

Density (kg/m3) 1046.14 ± 0.12aA 1043.25 ± 0.32a 1043.83 ± 0.93a

RI PT

Table 2. Physicochemical properties of fortified yogurt at 28 days of storage Time Acidity Syneresis Moisture Samples pH (days) (g/100mL) (g/100mL) (g/100g) aA bA cC 0 4.68 ± 0.01 0.86 ± 0.01 43.51 ± 0.01 84.02 ± 0.03aA 14 4.65 ± 0.03b 0.88 ± 0.01a 48.76 ± 0.01b 83.73 ± 0.09a Control c 28 4.43 ± 0.01 0.89 ± 0.01a 52.72 ± 0.01ª 84.00 ± 0.02a 4.65 ± 0.01aA 4.62 ± 0.02b 4.41 ± 0.01c

0.87 ± 0.01bA 42.10 ± 0.01cC 83.90 ± 0.01aA 1045.32 ± 0.52aA 0.88 ± 0.01a 47.75 ± 0.01b 83.75 ± 0.01a 1053.23 ± 0.83a 0.89 ± 0.01a 51.54 ± 0.01ª 83.90 ± 0.08a 1046.89 ± 0.59a

Ca15N

0 14 28

4.65 ± 0.01aA 4.60 ± 0.02b 4.33 ± 0.01c

0.87 ± 0.01bA 42.33 ± 0.01cC 83.90 ± 0.02aA 1049.66 ± 0.18aA 0.87 ± 0.01b 50.80 ± 0.01b 83.59 ± 0.07a 1054.90 ± 0.74a 0.88 ± 0.01a 53.54 ± 0.01ª 84.00 ± 0.04a 1049.12 ± 0.80a

Ca30M

0 14 28

4.62 ± 0.01aA 4.63 ± 0.05b 4.50 ± 0.00c

0.86 ± 0.01bA 46.52 ± 0.01cB 83.50 ± 0.01aA 1047.84 ± 0.32aA 0.87 ± 0.01b 53.00 ± 0.01b 83.90 ± 0.01a 1049.56 ± 0.45a 0.88 ± 0.01a 56.72 ± 0.01ª 84.00 ± 0.01a 1051.29 ± 0.38a

FZ50N

0 14 28

4.77 ± 0.01aA 4.63 ± 0.07b 4.30 ± 0.01c

0.87 ± 0.01bA 43.00 ± 0.01cC 83.75 ± 0.02aA 1049.18 ± 0.99aA 0.88 ± 0.01b 48.51 ± 0.01b 83.90 ± 0.01a 1046.56 ± 0.76a 0.90 ± 0.01a 53.45 ± 0.01ª 84.00 ± 0.04a 1044.01 ± 0.08a

FZ25N

0 14 28

4.74 ± 0.01aA 4.60 ± 0.03b 4.44 ± 0.00c

0.87 ± 0.01bA 43.54 ± 0.01cC 83.81 ± 0.03aA 1048.81 ± 0.26aA 0.87 ± 0.01b 51.95 ± 0.01b 83.94 ± 0.01a 1043.22 ± 0.61a 0.88 ± 0.01a 53.40 ± 0.01ª 84.00 ± 0.01a 1046.96 ± 0.98a

FZ50M

0 14 28

4.71 ± 0.01aA 4.54 ± 0.03b 4.25 ± 0.01c

0.86 ± 0.01bA 55.39 ± 0.01cA 83.71 ± 0.01aA 1051.33 ± 0.92aA 0.86 ± 0.01b 52.45 ± 0.01b 83.94 ± 0.01a 1047.41 ± 0.54a 0.91 ± 0.01a 56.30 ± 0.01ª 84.00 ± 0.01a 1048.18 ± 0.09a

Zn50N

0 14 28

4.77 ± 0.01aA 4.53 ± 0.02b 4.32 ± 0.01c

0.86 ± 0.01bA 48.48 ± 0.01cB 83.67 ± 0.01aA 1049.44 ± 0.70aA 0.86 ± 0.01b 46.84 ± 0.01b 83.87 ± 0.01a 1050.44 ± 0.34a 0.89 ± 0.01a 47.88 ± 0.01ª 84.00 ± 0.01a 1042.39 ± 0.78a

Zn25N

0 14 28

4.68 ± 0.01aA 4.63 ± 0.03b 4.34 ± 0.01c

0.86 ± 0.01bA 40.93 ± 0.01cD 83.76 ± 0.00aA 1046.32 ± 0.19aA 0.88 ± 0.01b 46.08 ± 0.01b 83.80 ± 0.09a 1043.41 ± 0.05a 0.89 ± 0.01a 47.21 ± 0.01ª 83.94 ± 0.08a 1044.41 ± 0.47a

0 14 28

4.68 ± 0.01aA 4.53 ± 0.03b 4.30 ± 0.01c

0.86 ± 0.01aA 51.18 ± 0.01cA 83.62 ± 0.05aA 1045.24 ± 0.47aA 0.86 ± 0.01b 54.15 ± 0.01b 83.81 ± 0.08a 1043.22 ± 0.61a 0.90 ± 0.01c 54.44 ± 0.01ª 84.00 ± 0.08a 1047.41 ± 0.54a

M AN U

TE D

EP

AC C

Zn50M

SC

Ca30N

0 14 28

Average of three replicates. Values with different letters are significantly different (P<0.05). Lowercase letters = differences during storage, capital letters=differences between samples.

2

ACCEPTED MANUSCRIPT

Table 3. Parameters of color of fortified yogurts at 28 days of storage Time Samples L a* b* (days)

∆E

89.71 ± 0.41aA -4.02 ± 0.02cA 12.08 ± 0.00aA 0.00 ± 0.00c 88.27 ± 0.09b -2.19 ± 0.01b 11.31 ± 0.05b 2.28 ± 0.08b 82.31 ± 0.01c -1.57 ± 0.04a 9.76 ± 0.04c 4.17 ± 0.03a

Ca30N

0 14 28

90.12 ± 0.18aA -4.12 ± 0.02cA 11.68 ± 0.12aA 0.00 ± 0.00b 88.12 ± 0.02a -2.21 ± 0.01b 10.95 ± 0.01b 2.01 ± 0.41a 83.56 ± 0.01b -1.58 ± 0.13a 9.60 ± 0.01c 2.06 ± 0.76a

Ca15N

0 14 28

89.76 ± 0.25aA -4.17 ± 0.02cA 11.80 ± 0.04aA 0.00 ± 0.00b 87.69 ± 0.40b -2.17 ± 0.02b 10.84 ± 0.05b 2.14 ± 0.09a 82.77 ± 0.44c -1.87 ± 0.01a 9.47 ± 0.03c 3.38 ± 0.17a

Ca30M

0 14 28

89.93 ± 0.11aA -4.15 ± 0.04cA 11.74 ± 0.02aA 0.00 ± 0.00c 86.23 ± 0.05b -1.97 ± 0.01b 10.50 ± 0.01b 3.46 ± 0.23b 83.11 ± 0.14c -1.72 ± 0.06a 9.78 ± 0.01c 3.38 ± 0.22a

FZ50N

0 14 28

86.69 ± 0.28aB 0.78 ± 0.09cB 10.58 ± 0.02aB 0.00 ± 0.00b 84.44 ± 0.10b 0.31 ± 0.04b 8.38 ± 0.04b 2.93 ± 0.00a c a 78.96 ± 0.60 3.34 ± 0.04 7.41 ± 0.23c 5.51 ± 0.00a

FZ25N

0 14 28

85.91 ± 0.07aB -0.04 ± 0.01cB 10.87 ± 0.09aB 0.00 ± 0.00c 83.34 ± 0.10b 1.86 ± 0.02b 9.11 ± 0.01b 2.93 ± 0.23b c a 78.40 ± 0.26 2.12 ± 0.14 8.36 ± 0.10c 5.51 ± 0.16a

FZ50M

0 14 28

Zn50N

0 14 28

SC

M AN U

TE D

86.03 ± 0.12aB -0.01 ± 0.01bB 10.71 ± 0.02aB 0.00 ± 0.00c 83.49 ± 0.25b 0.50 ± 0.08ab 9.48 ± 0.08b 4.29 ± 0.23a 81.05 ± 0.11c 1.16 ± 0.05a 8.83 ± 0.05c 2.90 ± 0.20b

EP

89.79 ± 0.34aA -4.18 ± 0.00bA 11.76 ± 0.01aA 0.00 ± 0.00b 86.16 ± 0.08b -1.99 ± 0.10a 10.08 ± 0.04b 3.99 ± 0.20a 82.11 ± 0.19c -1.82 ± 0.06a 9.67 ± 0.00c 4.40 ± 0.07a

0 14 28

89.57 ± 0.13aA -4.17 ± 0.00cA 11.80 ± 0.03aA 0.00 ± 0.00b 86.41 ± 0.06b -1.79 ± 0.04b 10.27 ± 0.02b 4.27 ± 0.01a 83.48 ± 0.07c -1.87 ± 0.07a 9.59 ± 0.03c 3.17 ± 0.13a

0 14 28

90.44 ± 0.48aA -4.10 ± 0.02cA 11.83 ± 0.06aA 0.00 ± 0.00c 88.03 ± 0.14b -2.12 ± 0.01b 10.01 ± 0.02b 2.60 ± 0.08b 81.43 ± 0.12c -1.67 ± 0.02a 8.93 ± 0.04c 4.56 ± 0.10a

AC C

Zn25N

RI PT

Control

0 14 28

Zn50M

Average of three replicates. Values with different letters are significantly different (P<0.05). ∆E= changes during storage. Lowercase letters = differences during storage, capital letters=differences between samples.

3

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Table 4. Root-mean-square error of rheological models. Samples RMSEPL RMSEHB Control 1.03 0.12 Ca30N 1.36 0.12 Ca15N 1.79 0.4 Ca30M 2.1 0.28 FZ50N 2.57 0.07 FZ25N 2.76 0.07 FZ50M 2.85 0.11 Zn50N 1.53 0.07 Zn25N 1.93 0.03 Zn50M 2.22 0.02 Values of three replicates, PL=Power Law model, HB= Herschel-Bulkley model.

4

ACCEPTED MANUSCRIPT

Table 5. Rheological properties of fortified yogurts at 28 days of storage Time KPL KHB Samples nPL nHB n (days) (Pa·s ) (Pa·sn)

τ0 (Pa)

0.34 ± 0.01aA 0.34 ± 0.03a 0.35 ± 0.01a

5.53 ± 0.03aB 5.15 ± 0.09b 4.05 ± 0.02c

0.36 ± 0.01aA 0.35 ± 0.01a 0.32 ± 0.01a

5.39 ± 0.03aB 4.76 ± 0.09b 3.76 ± 0.02c

0.47 ± 0.02aA 0.42 ± 0.02a 0.30 ± 0.03a

Ca30N

0 14 28

0.21 ± 0.01aA 0.27 ± 0.02a 0.35 ± 0.01a

8.19 ± 0.01aA 5.94 ± 0.01b 4.62 ± 0.08c

0.22 ± 0.01aA 0.29 ± 0.01a 0.30 ± 0.01a

7.55 ± 0.01aA 6.21 ± 0.01b 4.31 ± 0.08c

0.57 ± 0.02aA 0.61 ± 0.03a 0.38 ± 0.09a

Ca15N

0 14 28

0.30 ± 0.01aA 0.34 ± 0.02a 0.49 ± 0.01a

5.97 ± 0.02aB 4.00 ± 0.07b 2.78 ± 0.04c

0.32 ± 0.01aA 036 ± 0.01a 0.37 ± 0.01a

5.53 ± 0.02aB 3.73 ± 0.07b 2.55 ± 0.04c

0.47 ± 0.08aA 0.31 ± 0.04a 0.26 ± 0.00a

Ca30M

0 14 28

0.31 ± 0.01aA 0.35 ± 0.05a 0.39 ± 0.00a

5.51 ± 0.01aB 4.00 ± 0.01b 3.45 ± 0.01b

0.33 ± 0.01aA 0.36 ± 0.01a 0.29 ± 0.01a

5.16 ± 0.01aB 3.71 ± 0.01b 3.24 ± 0.01c

0.43 ± 0.02aA 0.32 ± 0.05a 0.35 ± 0.08a

0 14 28 0 14 28

0.27 ± 0.01aA 0.29 ± 0.07a 0.39 ± 0.01a 0.24 ± 0.01aA 0.35 ± 0.03a 0.41 ± 0.00a

5.41 ± 0.02aB 5.73 ± 0.01a 2.55 ± 0.04b 5.15 ± 0.03aB 3.72 ± 0.01b 2.88 ± 0.01c

0.28 ± 0.01aA 0.30 ± 0.01a 0.29 ± 0.01a 0.25 ± 0.01aA 0.37 ± 0.01a 0.38 ± 0.01a

5.01 ± 0.02aB 5.32 ± 0.01ab 2.44 ± 0.04b 6.19 ± 0.03aB 3.47 ± 0.01b 2.73 ± 0.01c

0.42 ± 0.09aA 0.35 ± 0.06a 0.29 ± 0.08a 0.35 ± 0.06aA 0.28 ± 0.01a 0.17 ± 0.08a

FZ50M

0 14 28

0.30 ± 0.01aA 0.32 ± 0.03a 0.34 ± 0.01a

6.06 ± 0.01aB 4.42 ± 0.01b 3.83 ± 0.01c

0.32 ± 0.01aA 0.34 ± 0.01a 0.23 ± 0.01a

5.58 ± 0.01aB 4.11 ± 0.01b 2.68 ± 0.01c

0.50 ± 0.02aA 0.33 ± 0.04a 0.19 ± 0.09a

Zn50N

0 14 28

0.26 ± 0.01aA 0.32 ± 0.02a 0.34 ± 0.01a

7.37 ± 0.01aA 4.89 ± 0.01b 3.80 ± 0.01c

0.28 ± 0.01aA 0.33 ± 0.01a 0.35 ± 0.01a

6.80 ± 0.01aA 4.93 ± 0.01b 3.58 ± 0.01c

0.60 ± 0.07aA 0.39 ± 0.04a 0.40 ± 0.08a

Zn25N

0 14 28

0.26 ± 0.01aA 0.33 ± 0.03a 0.36 ± 0.01a

0.28 ± 0.01aA 0.34 ± 0.01a 0.35 ± 0.01a

6.47 ± 0.00aB 4.49 ± 0.09b 4.00 ± 0.08c

0.31 ± 0.09aA 0.32 ± 0.05a 0.35 ± 0.07a

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FZ25N

6.31 ± 0.00aB 4.18 ± 0.09b 4.27 ± 0.08b

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FZ50N

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Control

0 14 28

0 0.20 ± 0.01aA 6.79 ± 0.05aB 0.22 ± 0.01aA 6.15 ± 0.05aB 0.57 ± 0.07aA 14 0.31 ± 0.03a 4.45 ± 0.08b 0.32 ± 0.01a 4.13 ± 0.08b 0.34 ± 0.01a Zn50M 28 0.37 ± 0.01a 3.39 ± 0.08c 0.29 ± 0.01a 3.21 ± 0.08c 0.30 ± 0.04a Average of three replicates. Values with different letters are significantly different (P<0.05). τ0 = Yield stress, n=flow index, K=consistency coefficient, PL=Power Law model, HB= HerschelBulkley model. Lowercase letters = differences during storage, capital letters=differences between samples.

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ACCEPTED MANUSCRIPT

Ca30M

0.93 ± 0.01aA 0.86 ± 0.02b 0.81 ± 0.01c

-0.65 ± 0.01aA -0.55 ± 0.01b -0.44 ± 0.01c

0 14 28 0 14 28

0.63 ± 0.01aC 0.60 ± 0.02b 0.58 ± 0.01c 0.60 ± 0.01aC 0.58 ± 0.05b 0.55 ± 0.00c

-0.57 ± 0.01aB -0-54 ± 0.01b -0.53 ± 0.01b -0.46 ± 0.01aC -0.43 ± 0.01b -0-40 ± 0.01c

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Ca15N

0 14 28

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Ca30N

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Table 6. Textural properties of fortified yogurts at 28 days of storage Firmness Cohesiveness Samples Time (days) (N) (dimensionless) 0 0.60 ± 0.01aC -0.42 ± 0.01aC b 14 0.56 ± 0.03 -0.41 ± 0.01a Control 28 0.55 ± 0.01c -0.39 ± 0.01b

0.60 ± 0.01aC 0.58 ± 0.07b 0.56 ± 0.01c

-0.43 ± 0.01aC -0.41 ± 0.01a -0.39 ± 0.01b

0.62 ± 0.01aC 0.60± 0.03b 0.58 ± 0.00c

-0.43 ± 0.01aC -0.40 ± 0.01b -0.32 ± 0.01c

FZ25N

0 14 28

FZ50M

0 14 28

0.60 ± 0.01aC 0.59 ± 0.03b 0.55 ± 0.01c

-0.39 ± 0.01aD -0.37 ± 0.01a -0.33 ± 0.01b

0 14 28

0.72 ± 0.01aB 0.69 ± 0.02b 0.68 ± 0.01c

-0.53 ± 0.01aB -0.45 ± 0.01b -0.38 ± 0.01c

0 14 28

0.66 ± 0.01aC 0.63 ± 0.03b 0.61 ± 0.01c

-0.47 ± 0.01aC -0.43 ± 0.01b -0.40 ± 0.01c

0 14 28

0.63 ± 0.01aC 0.61 ± 0.03b 0.58 ± 0.01c

-0.48± 0.01aBC -0.45 ± 0.01b -0.44 ± 0.01b

EP

Zn50N

TE D

FZ50N

0 14 28

AC C

Zn25N

Zn50M

Average of three replicates. Values with different letters are significantly different (P<0.05). Lowercase letters= differences during storage, capital letters= differences between samples.

6

ACCEPTED MANUSCRIPT

Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties.

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Figures

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ACCEPTED MANUSCRIPT

Figure 1. Rheograms of fortified yogurts: FZ50N (●), FZ25N (▲), FZ50M (▼), Ca15N (◄),

AC C

Ca15N (►), Ca30M (♦), Zn50N (□), Zn25N (+), Zn50M (*) and control (■), at 0 (a) and 28 days (b).

ACCEPTED MANUSCRIPT Fortification of yogurt with nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties. Highlights

Fortification of a dairy products with minerals.

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Nanoparticles and micro-minerals were incorporated to yogurt.

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Nanoparticles fortification improve syneresis compared to micro-minerals.

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Nanoparticles fortification does not alter the flow properties of yogurt.

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Nanoparticles fortification enhances the consistency and firmness of yogurt.

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