Blends of Pereskia aculeata Miller mucilage, guar gum, and gum Arabic added to fermented milk beverages

Accepted Manuscript Blends of Pereskia aculeata Miller mucilage, guar gum, and gum Arabic added to fermented milk beverages Tatiana Nunes Amaral, Luciana Affonso Junqueira, Mônica Elisabeth Torres Prado, Marcelo Angelo Cirillo, Luiz Ronaldo de Abreu, Fabiano Freire Costa, Jaime Vilela de Resende PII:

S0268-005X(17)31530-8

DOI:

10.1016/j.foodhyd.2018.01.009

Reference:

FOOHYD 4223

To appear in:

Food Hydrocolloids

Received Date: 6 September 2017 Revised Date:

15 December 2017

Accepted Date: 8 January 2018

Please cite this article as: Amaral, T.N., Junqueira, L.A., Torres Prado, Mô.Elisabeth., Cirillo, M.A., Ronaldo de Abreu, L., Costa, F.F., Vilela de Resende, J., Blends of Pereskia aculeata Miller mucilage, guar gum, and gum Arabic added to fermented milk beverages, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.01.009. 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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Blends of Pereskia aculeata Miller mucilage, guar gum, and gum arabic added to

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fermented milk beverages

3 4 Tatiana Nunes Amarala, Luciana Affonso Junqueiraa, Mônica Elisabeth Torres Pradoa, Marcelo

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Angelo Cirillob, Luiz Ronaldo de Abreua , Fabiano Freire Costac , Jaime Vilela de Resendea*

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a

Federal University of Lavras, Department of Food Science, Laboratory of Food Refrigeration, P.O.

Box 3037, 37200-000 Lavras, Minas Gerais, Brazil

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Minas Gerais, Brazil

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Gerais, Brazil

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Federal University of Lavras, Departament of Exact Sciences, P.O. Box 3037, 37200-000 Lavras,

Federal University of Juiz de Fora, College of Pharmacy, P.O. Box 36036-330. Juiz de Fora, Minas

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* Corresponding author: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

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Fermented milk products can be produced with hydrocolloids, such as guar gum (GG) and

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gum arabic (GA), to maintain quality during shelflife and to replace fat. Because of increasing

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demand for new hydrocolloids, Pereskia aculeata Miller (ora-pro-nobis; OPN) mucilage

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extract was prepared and may be a potential additive (gelling agent and/or emulsifier). One

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mixture design was used in model solutions, composed of OPN mucilage, GA, GG, sucrose,

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sodium chloride, and deionized water. The aim was to test rheological properties for all the

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treatments, and according to the results, to identify a suitable hydrocolloid blend to be added

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to fermented milk beverages (FMBs). The highest values of apparent viscosity of the model

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solutions were observed in the range where the hydrocolloid mix was composed of 70% OPN

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mucilage, 0% GA, and 30% GG, resulting in 0.5–0.7 Pa⋅s. Sucrose showed a greater influence

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on apparent viscosity than sodium chloride did. The effects of apparent viscosity, G’, and G”

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were discussed, and the model solutions revealed pseudoplastic behavior with a good fit to the

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power law rheological model. FMB samples appeared to conform to the power law,

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pseudoplastic and thixotropic behavior, and predominant elastic behavior (G’>G”). Addition

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of hydrocolloid blends increased pH, protein content, apparent viscosity, firmness, and

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adhesiveness and reduced syneresis of the FMB. Fat content influenced the texture

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parameters, and the microstructure was visualized and supported the results. Application of

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hydrocolloid blends containing OPN mucilage seems to be worthwhile and increases the

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protein content of FMBs.

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Keywords: Fermented milk beverage, Microstructure, Mixture design, Syneresis, Texture

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1. Introduction Fermented milk products are obtained by decreasing pH and via coagulation of milk

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with or without addition of other foods and dairy products. Lactic fermentation is driven by

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specific microbial species, which should be viable, active, and abundant in the final product

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during its shelflife (MAPA, 2007). Gel formation is the most important functional property of

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fermented dairy products, and the rheological characteristics of this gel are affected by the

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selected starter culture (Casarotti, Monteiro, Moretti, & Penna, 2014). Stabilizers such as

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hydrocolloids are commonly used in cultured products to control texture and to reduce whey

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separation. HM-pectin is widely used to avoid the flocculation of milk proteins and

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subsequent macroscopic whey separation. When the pH decreases there are interactions as

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function of charges between HM-pectin and casein micelle aggregates. HM-pectin adsorbs via

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electrostatic interactions in acidified milk systems where the adsorption takes place at or

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below pH 5.0 (Tromp, Kruif, van Eijk & Rolin, 2004; Jensen, Rolin & Ipsen, 2010). Lower-

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fat versions of cultured products usually need careful selection of stabilizers to restore the

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creaminess and other attributes of the full-fat product (Lucey, 2004).

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Guar gum (GG) is a hydrocolloid made from Cyamopsis tetragonoloba seeds. It

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essentially consists of galactose and mannose forming a complex carbohydrate polymer, and

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the industrial application is possible because of GG’s ability to form hydrogen bonding with

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water molecules; it is chiefly used as a thickener and stabilizer (Mudgil, Barak, & Khatkar,

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2014). Another hydrocolloid widely used in the industry is gum arabic (GA), an edible, dried,

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gummy exudate from the stems and branches of Acacia senegal and A. seyal. It is rich in

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nonviscous soluble fiber and serves as a stabilizer, thickening agent, and emulsifier (Ali,

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Ziada, & Blunden, 2009). GG and GA are both popular in the formulations of fermented dairy

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beverages.

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ACCEPTED MANUSCRIPT According to the increasing demand for hydrocolloids with specific functionality,

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finding new sources of hydrocolloids with appropriate properties is an active area of research

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(Salehi, Kashaninejad, Tadayyon, & Arabameri, 2015). Lima Junior et al. (2013) developed a

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process that allows for extraction of hydrocolloids from Pereskia aculeata Miller leaves. This

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plant belongs to the Cactaceae family and is commonly known in Brazil as ora-pro-nobis

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(OPN). Protein content of the leaves has been reported to be as high as in other vegetables,

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and they contain a high concentration of total dietary fiber and considerable amounts of

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carbohydrates and minerals (calcium, magnesium, manganese, and zinc) (Pinto & Scio, 2014;

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Takeiti, Antonio, Motta, Collares-Queiroz, & Park, 2009). The polysaccharide complexes of

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P. aculeata Miller leaves are highly ramified and contain arabinofuranose, arabinopyranose,

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galactopyranose, galactopyranosyl, uronic acid, and rhamnopyranose units (Sierakowski,

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Gorin, Reicher, & Corrêa, 1990). Recently, data on the chemical structure of polysaccharides

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from P. aculeata—evaluated by nondestructive methods such as nuclear magnetic resonance

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(NMR) spectroscopy—were published by Martin, Freitas, Sassaki, Evangelista, and

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Sierakowski (2017). The authors concluded that the mucilage of P. aculeata contains type I

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arabinogalactan as the main component, along with partially esterified galacturonic acid and

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fucose units. A study by Agostini-Costa, Pessoa, Gomes, and Silva (2014) indicated that the

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leaves of P. aculeata can serve as a source of antioxidants and provitamin A with a great

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potential to diversify and enrich the human diet.

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OPN proved to be an alternative source of mucilage, showing properties that make it

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useful in the industry as a thickener, gelling agent, and/or emulsifier. These findings have

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been confirmed by a linear increase in viscosity as a function of the concentration of the gum

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produced by reconstitution of the powder as well as by the high emulsion formation capacity

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and thermal stability of this emulsion at different temperatures (Conceição, Junqueira, Guedes

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Silva, Prado, & de Resende, 2014; Lima Junior et al., 2013). The macromolecular profile

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stabilizer (Lima Junior et al., 2013; Martin et al., 2017). The thickening properties and

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viscoelastic behavior of hydrocolloids in solution can be significantly affected by variables

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such as shear rate and time, compound concentrations, temperature, pressure, ionic strength,

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and pH. The analysis of individual or combined effects of these factors is important,

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especially when they will be used to modify food texture and for the design, evaluation, and

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modeling of processes (Amid & Mirhosseini, 2012; Capitani et al., 2015; Karazhiyan et al.,

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2009).

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Rheological behavior and interactions between hydrocolloids and other main

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components in food formulations are researched to explore the microstructure and to

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characterize thickening systems. The use of two or more gums in the formulation of a product

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is frequent in the food industry owing to the synergistic effects of combined use. It may result

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in an improvement of the product quality and provide economic benefits by decreasing the

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concentration of gum in the formulation. Given that apparent viscosity and physical stability

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of a food formulation may be modified by sugars and salts, at concentrations higher than

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those used for hydrocolloids, the research on these mixtures is justified (Chenlo, Moreira, &

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Silva, 2011). The aims of the present work were to evaluate the rheological properties of

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hydrocolloid blends (OPN mucilage, GA, and GG), sucrose, and sodium chloride in model

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solutions via mixture design as well as to verify the effects of the hydrocolloid-blend addition

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on

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microstructure of fermented milk beverages (FMBs).

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the physicochemical

properties,

rheological

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2. Materials and methods

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behavior,

syneresis,

texture,

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2.1. Extraction of OPN mucilage The process of extraction of OPN leaf mucilage was adapted from the procedure

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proposed by Lima Junior et al. (2013), as shown in Figure 1. Fresh OPN leaves were obtained

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in the municipality of Lavras/MG, Brazil and were harvested and transported to the

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laboratory. There, the leaves were selected, rinsed, weighed, and packed in labeled

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polyethylene bags. The packaged material was stored in a freezer (−18°C) until the mucilage

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extraction.

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Extraction 1 consisted of homogenization of the leaves in an industrial blender

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(MetvisaLG10, São Paulo, Brazil) with boiling water and subsequent extraction in a

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thermostatic water bath (Solab SL 150, Piracicaba/SP, Brazil) at 75 ± 1°C for 6 h. The

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material resulting from Extraction 1 was subjected to Extraction 2: pressing in a hydraulic

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press (Tecnal TE 058, Campinas, Brazil). The liquid fractions derived from Extractions 1 and

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2 were subjected to Filtering 1. This step was carried out by filtering the material with

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vacuum (dual-stage pump) in a Buchner funnel with organza fabric as a filter element, thus

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generating Filtrate 1.

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The latter was loaded on a fixed bed column (1.00 m height and 0.11 m diameter)

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containing activated carbon (Dinâmica Química, 1–2 mm) to remove insoluble solids and

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pigments (Filtering 2 phase). This process generated Filtrate 2, which was processed by

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precipitation with 95% ethanol (VETEC, PA ACS, Brazil) at a ratio of 3:1 of alcohol to

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Filtrate 2. The precipitate was centrifuged (SP Labor SP701, Presidente Prudente/SP, Brazil)

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(12 min, 6000 rpm, 4677 × g) for maximal removal of alcohol and dried in a freeze-drier

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(Edwards, mod. L4KR, São Paulo/SP, Brazil). The dry material was ground up in a ball mill

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(Marconi MA350, Piracicaba/SP, Brazil) for 1 min and stored away from light and moisture.

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The ethyl alcohol used at these steps was recovered by distillation and reused for

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precipitation.

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2.2. Preparation of model solutions The model solutions were prepared by dissolving the solution components (in a

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magnetic stirrer; 30 min, 75°C): deionized water, sucrose (Isofar), sodium chloride (Sigma-

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Aldrich), OPN mucilage, GA (Synth), and GG (Sigma-Aldrich) according to the experimental

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design (section 2.7). The solutions were kept in a thermostatic cabinet (Eletrolab, EL202, São

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Paulo/SP, Brazil) at 4°C for 24 h for complete hydration. Sodium chloride (NaCl) and sucrose

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were chosen as components of the solutions because they are the most abundant low-

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molecular-weight taste determinants (Antipova & Semenova, 1995).

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2.3. Preparation of FMBs

FMBs were prepared from whole or skim milk using the starter culture, composed of

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Lactobacillus acidophilus, Bifidobacterium, and Streptococcus. thermophilus (Bio Rich, Chr.

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Hansen), with sucrose (Isofar) and with or without addition of the hydrocolloid blends. The

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formulas of the samples were prepared according to the experimental design (section 2.9), and

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composition of the hydrocolloid blends was determined in preliminary tests using model

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solutions. The FMB production flowchart is shown in Figure 2.

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The two types of milk were first heat-treated and cooled. The corresponding sucrose

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and hydrocolloid mix were added and incubated overnight in a thermostatic cabinet at 4°C

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(Eletrolab, EL202, São Paulo/SP, Brazil) to complete the hydration of the systems. The milk

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solutions were heat-treated to minimize the presence of undesirable microorganisms from the

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added ingredients and were cooled down to 42°C. At that temperature, the starter culture was

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added, and the samples were incubated in an oven at 42°C for 6 h. After aging, the gel

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structure of the samples was broken with slight agitation and incubation overnight at 4°C until

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the analysis.

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2.4. Physicochemical analysis: milk Milk samples were characterized in terms of density (g⋅L−1) and content (%) of fat,

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lactose, solids, and proteins by means of a LactoScan ultrasonic milk analyzer (model

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G0041P, Bulgaria). The pH levels in the FMB samples were determined using a

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potentiometer (Schott Handylab). The FMB protein content was determined by the Kjeldahl

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method (AOAC Official Method 984.13 [A-D], 2006, using the formula 6.25 × nitrogen

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value).

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2.5. Rheological analysis

These assays were conducted on a rheometer, HAAKE RheoStress 6000 (Thermo

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Scientific, Karlsruhe, Germany) coupled to a temperature controller HAAKE UTM (Thermo

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Scientific). The model solutions were analyzed using parallel plate geometry (34.997 mm

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diameter), GAP 1 mm at 20°C. Characterization of the rheological behavior was performed

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by means of flow curves, subjecting the samples to a continuous ramp shear rate ( ) between

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0 and 300 s−1 for 2 min with 100 readings of shear stress ( ). For construction of the flow

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curves, samples were first stabilized for 3 min at 20°C and subjected to thixotropy breaking,

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by applying a continuous ramp shear rate ( ) between 0 and 300 s−1 for 2 min for ramping

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and the same period was used for a gradual decrease from 300 to 0 s−1.

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Oscillatory tests were performed to study the viscoelastic properties of the samples.

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Determination of the linear viscoelastic range was carried out by subjecting the samples to

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shear stress between 0.001 and 100 Pa with a fixed frequency of 1 Hz at 20°C. The frequency

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sweep curves were generated assuming a value of shear stress within the linear viscoelastic

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range, with the frequency varying from 0.01 to 10 Hz at 20°C. FMB samples were analyzed using cone plate geometry (34.998 mm diameter, 2°),

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GAP 0.105mm at 10°C (Braga & Cunha, 2004). Flow curves were obtained by subjecting the

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samples to a continuous shear rate ( ) ramp (0 to 300 s−1 for 2 min, 100 readings). For

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generation of the flow curves, the samples were first stabilized for 3 min at 10°C and

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subjected to thixotropy breaking (0–300 s−1 and 300–0 s−1 ramps).

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The oscillatory tests were as follows: determination of the linear viscoelastic range

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(shear stress between 0.001 and 100 Pa, 1 Hz, 10°C) and frequency sweep curves, assuming a

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shear stress value within the linear viscoelastic range and frequency range from 0.01 to 10 Hz

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at 10°C.

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Data obtained on the flow curves were fitted to the Newton (Equation 1) and power

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law (Equation 2) rheological models, generating responses to the fluid classification and the

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adjusted model parameters.

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=

∙

=

∙

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(2)

where

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coefficient, and n is the flow behavior index.

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is shear stress (Pa),

is shear rate (s−1), µ is viscosity (Pa⋅s), K is the consistency

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(1)

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Thixotropy values were calculated from the difference between the curve areas of rise

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and fall prior to construction of a flow curve. The apparent viscosity values were calculated

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from the shear stress data point at 100 s−1, related to the value observed in the fluid in

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industrial pipes (Steffe, 2006). In the frequency sweep curves, we examined the behavior of

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G’ and G” and studied the effect plots of G’ and G” at 1-Hz frequency.

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2.6. Syneresis measurements in the FMB Syneresis in FMBs with and without hydrocolloid blends was measured by the

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drainage method and centrifugation method according to Harwalkar and Kalab (1986). In the

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drainage method, FMB was drained via a funnel with a stainless-steel screen (120 mesh), and

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the whey volume separated in a calibrated cylinder was measured at 5-min intervals for 60

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min.

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In the centrifugation method, 15-mL aliquots of FMB were placed in graduated plastic

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tubes and were centrifuged at 6 °C for 10 min, with 60 s of acceleration and 60 s of braking.

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The speeds of centrifugation were 480, 1900, 2770, 3400, and 3920 rpm (30, 500, 1000,

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1500,and 2000 × g, respectively). The supernatant volume after centrifugation was quantified,

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and syneresis (S) was calculated by means of equation (3):

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× 100

(3)

where Vs is the supernatant volume, and Vi is the initial volume.

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%) =

2.7. Instrumental texture analysis

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A texture profile analysis (TPA) was performed using a texturometer (TA-XT2i,

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Texture Tech. Corp., Scarsdale, USA) and the Texture Expert software (v.1.22, 1999, Stable

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Micro Systems, Godalming, UK) (Santillán-Urquiza, Méndez-Rojas, & Vélez-Ruiz, 2017).

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The double compression force (N) in all the samples of FMB was applied using a cylindrical

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probe of 20 mm in diameter, descending at a speed of 1.0 mm/s, and reaching a depth of 15

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mm with cycle intervals of 5 s. Firmness and adhesiveness were the evaluated parameters.

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2.8. Microstructural analysis The microstructural analyses of FMB samples were carried out by scanning electron

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microscopy (SEM) and fluoresecence microscopy. The FMB samples were freeze-dried

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(Edwards,L4KR, São Paulo/SP, Brazil) (Espírito-Santo et al., 2013), their dried samples were

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attached with double-sided carbon tape to aluminum supports (stubs), and the samples were

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sputter-coated in vacuum with a thin film of metallic gold using a Bal-Tec model MED 020

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evaporator (Balzers, Liechtenstein). A scanning electron microscope (JEOL JSM 6360 LV,

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Akishima, Japan) was used at accelerating voltage of 10 kV to obtain digital images at varied

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magnification. The images were processed in the Corel Draw 14 Photo Paint software.

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Fluorescence microscopy was carried out under an Axio Observer Z1 microscope

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(Carl Zeiss Microimaging GmbH, Göttingen, Germany), and the images were taken using the

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Zeiss AxioVision 4.6 Image Program. The fluorochrome was Red Nile (Sigma-Aldrich,

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N3013-100 mg) that was prepared according to technical information (ATTBioquest, 2012).

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2.9. Experimental design and statistical analysis For model solutions, mixing tests (mixture design) were developed involving as

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variables the hydrocolloid OPN mucilage, GG, and GA. Three hydrocolloid blend systems

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were proposed and tested (Table 1). The blends of OPN mucilage, GG, and GA were prepared

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according to the mixture design with 9 data points (Figure 3). The experimental domain of

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this research consisted of different proportions of each component: A (OPN mucilage) and B

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(GA) between 0 and 100%, and C (GG) between 0 and 33%. GG had its ratio restricted to

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33% for better evaluation of the other studied gums. The component proportions of the blends

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are shown in Table 2. All the samples were prepared in three independent replicates to allow

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for error estimation. The quadratic model defined in Equation 4 was adjusted according to the

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proposed design (Statistica StatSoft 8). Graphs were built based on this model to study the

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relation between the effects of the components and the influence of the responses.

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+ ∑ ∑ "!

∗ !

(4)

!

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Where

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)=∑

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is the value predicted by the quadratic model, and denotes the estimate of

the parameters relating to the principal effects and interactions, i = 1 component, q = 3 (total

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number of components), and j is the number of data points.

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For application to FMB, a completely randomized factorial design was employed for

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statistical analysis and implemented in the SAS statistical software. All the experiments were

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conducted at least in duplicate, and the data were presented as a mean of each experiment.

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The t-test and Scott–Knott test were applied to compare means, and the differences were

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considered significant at Pvalues<0.05. Curves, rheological parameters of fitted models,

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adjusted values (coefficients of determination [R2] and root mean square error [RMSE]) were

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obtained by means of the SAS statistical software. The formulations of the FMB samples are

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shown in Table 3.

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3. Results and discussion

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3.1. Model solutions

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3.1.1. Mixture experiment: rheological responses

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The general purpose in the mixture design is to make it possible to estimate the

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properties of a multicomponent system after a limited number of observations. The theory of

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experimentation and modeling for mixture design encompasses designs and suitable

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polynomial regression to the specific tests involving proportions of components

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(Nepomucena, Silva, & Cirillo, 2013). Methods for experiments with mixtures represent an

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area of applied statistics, important in food science and the industry because all foods are

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mixtures of a number of ingredients (Bjerke, Næs, & Ellekjær, 2000). The experimental values that we obtained for apparent viscosity (shear rate at 100 s−1),

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storage modulus (G’), and loss modulus (G”) were adjusted by means of Equation 4, and the

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respective parameters are shown in Table 4. All systems showed R2>0.95 for the three

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responses. Significant components made an impressive contribution to the prediction of the

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respective model.

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The application of equations using the parameters presented in Table 4 resulted in the

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contour line graphs shown in Figures 4A1-C1. The curves show the apparent viscosity

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behavior of each system. System 1 (Figures 4A1-A2) comprises sucrose, NaCl, and the

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hydrocolloid mix, with maximum values of viscosity similar to those in system 2 (Figures

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4B1-B2), which consisted of NaCl. System 3 (Figures 4C1-4C2, without sucrose) even with

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behavior similar to that of other systems had a smaller increase in apparent viscosity of the

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samples, with maximum value ~0.5 Pa⋅s. The highest values of apparent viscosity in the

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model solutions correspond to the range of composition where the hydrocolloid mix was

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composed of 70% OPN mucilage, 0% GA and 30% GG, resulting in 0.5–0.7 Pa⋅s.

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Effect plots of components for G’ and G” are shown in Figure 5. The effect behaviors

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of G’ and G” in the same system were similar, varying among the three systems. OPN

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mucilage and GA had similar effects on system 1 (Figures 5A1-5A2), decreasing until the

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midpoint, then stabilizing, whereas GG yielded a rapid increase until the midpoint. System 3

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(Figures 5C1-5C2) manifested similar behavior, but GA yielded a greater reduction gradient

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than OPN mucilage did. System 2 (Figures 5B1-5B2), without NaCl, behaved differently

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from the other systems. GG caused a rapid linear increase related to the centroid point for G’,

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and a similar behavior was seen for G”. OPN mucilage had a tendency for linear behavior

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with a smaller decrease than that of GA.

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3.1.2. Rheological behavior The rheological behavior of sucrose solutions is Newtonian at any concentration

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(Saggin & Coupland, 2004), but with the addition of hydrocolloids, the system can change the

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behavior. In all three systems studied, the samples containing GG had 99% adjustment to the

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rheological model of power law and showed pseudoplastic behavior. According to Galmarini,

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Baeza, Sanchez, Zamora, and Chirife (2011), the addition of GG at 0.1 g per 100g to a sugar

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solution results in pseudoplastic behavior as observed for GG solutions in the present study.

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The samples composed only of GA as a hydrocolloid revealed Newtonian behavior

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with adjustments above 75% in all three systems. GA is a biopolymer that contains

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amphiphilic compounds (proteins and polysaccharide–protein complexes) and the presence of

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solutes (sucrose and/or NaCl) changes GA behavior in an aqueous solution to the one that is

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non-Newtonian according to Gómez-Díaz, Navaza, and Quintáns-Riveiro (2008). The

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samples that were composed only of OPN mucilage as a hydrocolloid showed pseudoplastic

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behavior (R2 > 87%: power law) in systems 1 and 3. System 2, without NaCl, manifested

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Newtonian behavior (R2> 97%).

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The samples under study showed thixotropic behavior and did not differ significantly

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at 5% significance according to the Scott–Knott test (data not shown). The thixotropy is

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caused by the structural breakdown in a dispersion under shear stress. The thixotropy in

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heated protein suspensions can be attributed to the particle breakage or the breakdown of

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bonds such as disulfide, Van der Walls, ionic, and hydrophobic interactions between protein

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particles (Martin, 1993, Kolsoy & Kilic, 2004).

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3.2.Addition to FMB Judging by the results obtained with the model solutions (section 3.1), we selected the

343

hydrocolloid mixture composed of 2.19% OPN mucilage and 0.81% GG to be applied to the

344

FMB (Table3), constituting 3% of the whole formula. These values are within the optimal

345

ranges of apparent viscosity that were evaluated using the model systems (Figure 4).

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346 347

3.2.1.Physicochemical characterization milk

Whole milk and skim milk used for preparation of FMB were analyzed, and the results

349

are shown in Table 5. The two types of milk did not differ in pH values, proving that this

350

parameter did not affect the results of the FMB treatments. Lactose, nonfat solids, protein, fat

351

content (and therefore density) were significantly different. The main difference among the

352

samples was observed in fat content.

353

3.2.2.Rheological behavior of FMB

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354

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348

Flow curves of FMB samples were generated (Figure 6), and their rheological data

356

were adjusted to the power law model (Table 6). The samples showed a good fit (R2>94%)

357

and pseudoplastic behavior. This behavior was confirmed by the values of the consistency

358

coefficient greater than zero (K> 0) and index flow behavior values between zero and one (0

359


360

FW (abbreviations are defined in Table 3), followed by WH and SH, and smaller values of n

361

were detected in samples FWH and FSH. The addition of hydrocolloid blends resulted in an

362

increased consistency coefficient (K) in FMB and was accompanied by an increase in the

363

pseudoplasticity manifested in the decrease of the flow behavior index (n) (Kolsoy & Kilic,

364

2004). Figure 6(B) shows the apparent viscosity as function of shear rate for all the

365

treatments.

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15

ACCEPTED MANUSCRIPT The pH values and protein content of the samples are shown in Table 7. Using these

367

values, it is possible to confirm the correct fermentation with the inoculated samples by

368

increasing acidity of the medium. The addition of hydrocolloids reduced acidity of the

369

samples. Bacterial fermentation converts lactose into lactic acid, which reduces pH of milk.

370

During milk acidification, pH decreases to values lower than 4.6 (pH = 4.6 is the isoelectric

371

point of casein). Gelation occurs at pH 5.2 to 5.4 for milk that has undergone thermal

372

treatment (Lee & Lucey, 2010). In table 7, it is shown that FMB had pH values below the

373

isoelectric point of casein for treatments without hydrocolloids (pH = 4.09 for FW and pH =

374

4.03 for FS) and above but close to the isoelectric point for samples with addition of

375

hydrocolloids (pH = 4.80 for FWH and pH = 5.22 for FSH). When pH of an FMB is close to

376

the isoelectric point (pH = 4.6), there is a reduction in the net negative charge of casein; this

377

change leads to a decrease in electrostatic repulsion between casein molecules (Lucey, Munro

378

& Singh, 1998; Lee & Lucey, 2010).The acidification process results in the formation of a

379

three-dimensional network of clusters and casein chains. The milk fat content did not

380

significantly influence pH in the FMB (P< 0.05).

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366

In Table 7, it is evident that protein content of FMB increased significantly (P< 0.05)

382

in samples with hydrocolloid blends. The increase can mainly be attributed to the added OPN

383

mucilage whose protein content was found to be 10% (w/w) by Lima Junior et al. (2013) after

384

drying the mucilage and 15% (w/w) by Martin et al. (2017) following another extraction

385

method.

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381

The apparent viscosity data at 100 s−1 are also presented in Table 7. FMB without the

387

addition of a hydrocolloid had lower apparent viscosity and did not differ from whole or skim

388

milk (P<0.05), followed by FWH and FSH and by the two samples without fermentation (SH

389

and WH). The sample with a higher apparent viscosity value was FSH.

16

ACCEPTED MANUSCRIPT The effect of mixtures of polysaccharides with proteins (GG and soy protein) on

391

apparent viscosity was observed by Galmarini et al. (2011). These authors proposed formation

392

of weak electrostatic interactions and hydrogen bonds, forming a network that allows for

393

greater water retention and results in a bigger increase in viscosity than that contributed by the

394

individual components of the solution.

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All the FMB samples yielded values of G’ greater than G” as presented in Table 7.

396

This phenomenon reflects the predominance of elastic behavior of the samples representing

397

the structural resistance contributing to the three-dimensional network. The interactions

398

among hydrocolloids enable the formation of an elastic gel that is related to the configuration

399

of these chains. In the gelation profiles during the gel formation, G’ values increase due to the

400

formation of additional bonds between protein particles during rearrangements in the protein

401

network (Groseberg & Khokhlov, 1994; Lee & Lucey, 2010). In the present study, fat content

402

influenced these rheological parameters.

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395

404

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403

3.2.3. Syneresis, texture, and microstructure

Figure 7A and B shows that the FMB samples without hydrocolloid blends were

406

susceptible to syneresis during application of the two methods under study. Syneresis is the

407

separation of phases in a suspension or mixture, and the hydrocolloid blends were added to

408

the FMB for the purpose of increasing viscosity and decreasing susceptibility to syneresis

409

(Shellhass & Morris, 1985). Readers can see in Figure 7A and B and Table 7 that the effects

410

are due to pH values of the samples that are below 4.6, thereby contributing to casein

411

rearrangements and a water release, as observed by Santillan-Urquisa et al. (2017).

AC C

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412

Figure 8 suggests that the hydrocolloid blends influence the results on firmness and

413

adhesiveness of the FMB samples. The firmness (positive force peaks), which is the force

414

required to attain given deformation, and adhesiveness (area under the x-axis), which is a

17

ACCEPTED MANUSCRIPT measure of tensile strength for FMB samples, can be observed in the texture profile analysis

416

(TPA) depicted in Figure 8. The comparison between treatments indicates that the values of

417

firmness and adhesiveness are higher when the samples are prepared from skim milk. These

418

parameters are affected by the structural arrangement of the protein network, which is

419

influenced by the composition of the systems.

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The firmness and adhesiveness of the FMB samples are not indicative of susceptibility

421

to syneresis and are influenced by the milk fat content. The results on the texture parameters

422

revealed that the determining factors that justify the syneresis are the pore sizes in the protein

423

network, which decrease after addition of the hydrocolloid blends. Figure 9A presents

424

photomicrographs of freeze-dried FMB samples without and with hydrocolloid blends. Figure

425

9A1 and A2 (without hydrocolloid blends: FW and FS) shows structures with larger cavities

426

left after sublimation of large ice crystals. The ice crystals that filled these cavities were

427

formed by available water and were released into the pores of the protein matrix. In samples

428

with hydrocolloid blends, the cavity sizes decreased (Figure 9B1 and B2, FWH and FSH,

429

respectively). According to Hawalkar and Kalab (1986), formation of large pores in a protein

430

network can be caused by an increase in the positive electrical charge of the casein micelles at

431

pH < 4.6. The increase in the positive electric charge reduces intermicellar interactions, which

432

result in the formation of an open (porous) structure that leads to syneresis.

M AN U

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The fat globules are associated with the casein micelles and hydrocolloid blends as

AC C

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SC

420

434

shown in the photomicrographs of fluorescent signals with a lipid marker (Figure 9C1–D2).

435

These photomicrographs show relative absence of fat globules in skim milk relative to whole

436

milk and explain the influence of fat content on the texture parameters of the systems. The

437

values of firmness and adhesiveness decreased in the samples of FMB prepared from whole

438

milk with or without the addition of hydrocolloid blends.

439

18

ACCEPTED MANUSCRIPT 440

4. Conclusions The highest values of apparent viscosity in the model solutions were observed when

442

the hydrocolloids mix was composed of 70% OPN mucilage, 0% GA, and 30% GG, resulting

443

in 0.5–0.7 Pa⋅s. Sucrose had greater effects than sodium chloride did on apparent viscosity.

444

Model solutions showed pseudoplastic behavior with a good fit to the power law model.

RI PT

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FMB samples revealed a good fit to the power law and to pseudoplastic and

446

thixotropic behavior, and showed predominant elastic behavior (G’ > G”). The application of

447

hydrocolloid blends increased apparent viscosity, pH, protein content, firmness, and

448

adhesiveness when compared with the treatments without these additives. Syneresis was

449

lower in the samples with hydrocolloid blends. Milk fat content influenced rheological

450

behavior and texture parameters. Addition of hydrocolloid blends containing OPN mucilage

451

to fermented milk proved to be a viable approach. Shelf life determination and

452

microbiological and sensorial analyses are the objects of research in progress.

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454

5. Acknowledgements

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453

The authors wish to thank the Fundação de Amparo à Pesquisa do Estado de Minas

456

Gerais (FAPEMIG - Brazil), Conselho Nacional de Desenvolvimento Científico e

457

Tecnológico (CNPq - Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível

458

Superior (CAPES - Brazil) for financial support for this research, technical support and

459

supplied equipment (UFMG Microscopy Center) and material donation (Verde Campo LTDA

460

and Gemacon Tech).

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461 462

6. References

463

Agostini-Costa, T. S., Pêssoa, G. K. A., Silva, D. B., Gomes, I. S. & Silva J. P. (2014). Carotenoid

464

composition of berries and leaves from Cactaceae - Pereskia sp.. Journal of Functional Foods. 11, 178-

465

184. 19

ACCEPTED MANUSCRIPT Ali, B. H., Ziada, A., & Blunden, G. (2009). Biological effects of gum arabic: A review of some recent

467

research. Food and Chemical Toxicology, 47(1), 1–8.

468

Amid, B. T., & Mirhosseini, H. (2012). Influence of different purification and drying methods on

469

rheological properties and viscoelastic behaviour of durian seed gum. Carbohydrate Polymers, 90(1),

470

452–461.

471

Antipova, A. S., & Semenova, M. G. (1995). Effect of sucrose on the thermodynamic incompatibility

472

of different biopolymers. Carbohydrate Polymers, 28(4), 359–365.

473

AOAC- Association of Official Analytical Chemists (2006). Official Methods of Analysis of the

474

Association of Official Agricultural Chemists. 18 ed. Washington, DC, USA.

475

ATTBioquest. (2012). Nile red * Ultra pure grade * Assay protocol with nile red.

476

Bjerke, F., Næs, T., & Ellekjær, M. R. (2000). An application of projection design in product

477

development. Chemometrics and Intelligent Laboratory Systems, 51(1), 23–36.

478

Braga, A. L. M., & Cunha, R. L. (2004). The effects of xanthan conformation and sucrose

479

concentration on the rheological properties of acidified sodium caseinate-xanthan gels. Food

480

Hydrocolloids, 18(6), 977–986.

481

Capitani, M. I., Corzo-Rios, L. J., Chel-Guerrero, L. A., Betancur-Ancona, D. A., Nolasco, S. M., &

482

Tomás, M. C. (2015). Rheological properties of aqueous dispersions of chia (Salvia hispanica L.)

483

mucilage. Journal of Food Engineering, 149, 70–77.

484

Casarotti, S. N., Monteiro, D. A., Moretti, M. M. S., & Penna, A. L. B. (2014). Influence of the

485

combination of probiotic cultures during fermentation and storage of fermented milk. Food Research

486

International, 59, 67–75.

487

Chenlo, F., Moreira, R., & Silva, C. (2011). Steady-shear flow of semidilute guar gum solutions with

488

sucrose, glucose and sodium chloride at different temperatures. Journal of Food Engineering, 107(2),

489

234–240.

490

Conceição, M. C., Junqueira, L. A., Guedes Silva, K. C., Prado, M. E. T., & de Resende, J. V. (2014).

491

Thermal and microstructural stability of a powdered gum derived from Pereskia aculeata Miller

492

leaves. Food Hydrocolloids, 40, 104–114.

AC C

EP

TE D

M AN U

SC

RI PT

466

20

ACCEPTED MANUSCRIPT Espírito-Santo, A. P., Lagazzo, A., Sousa, A. L. O. P., Perego, P., Converti, A., & Oliveira, M. N.

494

(2013). Rheology, spontaneous whey separation, microstructure and sensorial characteristics of

495

probiotic yoghurts enriched with passion fruit fiber. Food Research International, 50(1), 224–231.

496

Galmarini, M. V., Baeza, R., Sanchez, V., Zamora, M. C., & Chirife, J. (2011). Comparison of the

497

viscosity of trehalose and sucrose solutions at various temperatures: Effect of guar gum addition. LWT

498

- Food Science and Technology, 44(1), 186–190.

499

Gómez-Díaz, D., Navaza, J. M., & Quintáns-Riveiro, L. C. (2008). Intrinsic Viscosity and Flow

500

Behaviour of Arabic Gum Aqueous Solutions. International Journal of Food Properties, 11(4), 773–

501

780.

502

Groseberg, A. Y. U., & Khokhlov, A. R. (1994). Statistical Physics of macromolecules. American

503

Institute of Physics Press.

504

Harwalkar, V. R. & Kalab, M. (1986). Relation between microstructure and susceptibility to syneresis

505

in yoghurt made from reconstituted nonfat dry milk, Food Structure, 5(2), Article 13.

506

Jensen, S., Rolin, C. & Ipsen R. (2010). Stabilization of acidified skimmed milk with HM pectin. Food

507

Hydrocolloids, 24, 291-299.

508

Karazhiyan, H., Razavi, S. M. A., Phillips, G. O., Fang, Y., Al-Assaf, S., Nishinari, K., & Farhoosh,

509

R. (2009). Rheological properties of Lepidium sativum seed extract as a function of concentration,

510

temperature and time. Food Hydrocolloids, 23(8), 2062–2068.

511

Kolsoy, A. & Kilic, M. (2004). Use of hydrocolloids in textural stabilization of a yoghurt drink, ayran.

512

Food Hydrocolloids, 18, 593-600.

513

Lee, W. J. & Lucey, J. A. (2010). Formation and physical properties of yogurt. Asian-Australia

514

Journal of animal Science, 23(9), 1127-1136.

515

Lima Junior, F. A., Conceição, M. C., Vilela de Resende, J., Junqueira, L. A., Pereira, C. G., & Torres

516

Prado, M. E. (2013). Response surface methodology for optimization of the mucilage extraction

517

process from Pereskia aculeata Miller. Food Hydrocolloids, 33(1), 38–47.

518

Lucey, J. A. (2004). Cultured dairy products: An overview of their gelation and texture properties.

519

International Journal of Dairy Technology, 57(2-3), 77–84.

AC C

EP

TE D

M AN U

SC

RI PT

493

21

ACCEPTED MANUSCRIPT 520

Lucey, J. A., Munro, P. A. & Singh H. (1998). Rheological properties and microstructure of acid milk

521

gels as affected by fat content and heat treatment. Journal of Food Science, 63(4), 660-664.

522

MAPA.

523

doi:10.1017/CBO9781107415324.004

524

Martin, A. N. (1993). Physical pharmacy. London: Lea & Febiger.

525

Martin, A. A., Freitas, R. A., Sassaki, G. L., Evangelista, P. H. L. & Sierakowski, M. R. (2017).

526

Chemical structure and physical-chemical properties of mucilage from the leaves of Pereskia

527

aculeata. Food Hydrocolloids, 70, 20-28.

528

Mudgil, D., Barak, S., & Khatkar, B. S. (2014). Guar gum: processing, properties and food

529

applications—A Review. Journal of Food Science and Technology, 51(3), 409–418.

530

Nepomucena, T. M., Silva, A. M. da, & Cirillo, M. A. (2013). Modelos Ridge em planejamentos de

531

misturas: uma aplicação na extração de polpa de pequi. Quimica Nova, 36(1), 159–164.

532

Pinto, N. de C. C., & Scio, E. (2014). The Biological Activities and Chemical Composition of Pereskia

533

Species (Cactaceae)-A Review. Plant Foods for Human Nutrition, 69, 189–195.

534

Saggin, R., & Coupland, J. N. (2004). Rheology of xanthan/sucrose mixtures at ultrasonic frequencies.

535

Journal of Food Engineering, 65(1), 49–53.

536

Salehi, F., Kashaninejad, M., Tadayyon, A., & Arabameri, F. (2015). Modeling of extraction process

537

of crude polysaccharides from Basil seeds (Ocimum basilicum l.) as affected by process variables.

538

Journal of Food Science and Technology, 52, 5220–5227.

539

Santillan-Urquiza, E., Mendez-Rojas, M. A. & Velez-Ruiz, J. F. (2017). Fortification of yogurt with

540

nano and micro sized calcium, iron and zinc, effect on the physicochemical and rheological properties.

541

LWT - Food Science and Technology, 80, 462 - 469.

542

Schellhaass, S. M. & Morris, H. A. (1985). Rheological and Scanning Electron Microscopic

543

Examination of Skim Milk Gels. Food Structure, 4(2), Article 11.

544

Obtained by Fermenting With Ropy and Non-Ropy Strains of Lactic Acid Bacteria,"

545

Sierakowski, M.-R., Gorin, P. a. J., Reicher, F., & Corrêa, J. B. C. (1990). Location of O-acetyl groups

546

in the heteropolysaccharide of the cactus Pereskia aculeata. Carbohydrate Research, 201(2), 277–284.

normativa

n°

46,

23

de

outubro

de

2007.

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Instrução

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(2007).

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ACCEPTED MANUSCRIPT Steffe, J. F. (2006). Rheological Methods in food process engineering. Agricultural Engineering (Vol.

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23). East Lansing: Freeman Press. doi:10.1016/0260-8774(94)90090-6

549

Takeiti, C. Y., Antonio, G. C., Motta, E. M. P., Collares-Queiroz, F. P., & Park, K. J. (2009). Nutritive

550

evaluation of a non-conventional leafy vegetable (Pereskia aculeata Miller). International Journal of

551

Food Sciences and Nutrition, 60 Suppl 1(August), 148–160.

552

Tromp, R. H., Kruif, C. G. van Eijk & Rolin, C. (2004). On the mechanism of stabilization of

553

acidified milk drinks by pectin. Food Hydrocolloids, 18, 565-572.

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Figure legends

556

Figure 1. The process of extraction of OPN leaf mucilage.

557

Figure 2. The flowchart of fermented milk production.

558

Figure 3. Mixture design with nine coded data points. A: OPN mucilage, B: GA, and C: GG.

559

Figure 4. 1) Contour lines and 2) an effect plot of components versus apparent viscosity (Pa.s)

562

TE D

561

at 100 s−1. A) System 1, B) System 2, and C) System 3.

Figure 5. The effect plot of components for 1) G’ (Pa) at 1Hz and 2) G” (Pa) at 1Hz. A) System 1, B) System 2, and C) system 3.

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555

Figure 6. Flow curves (A) and apparent viscosity curves (B) as a function of the shear rate.

564

Figure 7. Syneresis of fermented milk beverages. A) Drainage method and; B) centrifugation

565

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563

method.

566

Figure 8. Texture profile analysis (TPA) of fermented milk beverages.

567

Figure 9. SEM (A and B) (50× and 2500× magnification) and fluorescence analysis using Red

568

Nile (C and D) (100× magnification) photomicrographs of the FMB samples: (A and

569

C) Fermented milk, (B and D) Fermented milk with hydrocolloid blends. (1) Whole

570

milk, (2) skim milk.

23

ACCEPTED MANUSCRIPT Table 1. Fixed systems for the mixture tests. Hydrocolloid

systems

mix (%)

System 1

3

System 2 System 3

Sucrose (%)

Sodium

Deionized

cloride (%)

water (%)

10

2

85

3

10

0

87

3

0

2

95

Table 2. Mixture design with nine ratios of hydrocolloid blends.

RI PT

Fixed

Component proportion

solution

(sum = 1)

SC

Model

GA

1

0.2088

0.7088

0.0825

2

0.0000

0.6700

0.3300

3

0.7088

0.2088

0.0825

4

0.0000

1.0000

0.0000

5

1.0000

0.0000

0.0000

6

0.4175

0.4175

0.1650

7

0.6700

0.0000

0.3300

8

0.5438

0.2088

0.2475

0.2088

0.5438

0.2475

0.4175

0.4175

0.1650

0.4175

0.4175

0.1650

10

AC C

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11

TE D

9

M AN U

OPN mucilage

1

GG

ACCEPTED MANUSCRIPT

Table 3. FMB formulations with hydrocolloid mixes. Milk

Milk (g)

type

Sucrose

Starter

Hydrocolloid Blends

(g)

culture (g)

(g) OPN

GG

RI PT

Sample W

89.964

10.000

0,036

2.190

0.810

SH

S

89.964

10.000

0.036

2.190

0.810

FW

W

89.964

10.000

0,036

0.000

0.000

FS

S

89.964

10.000

0,036

0.000

0.000

FWH

W

89.964

10.000

0,036

2.190

0.810

FSH

S

89.964

10.000

0,036

2.190

0.810

SC

WH

M AN U

WH: whole milk with hydrocolloids; SH: skim milk with hydrocolloids, FW: fermented whole milk; FS: fermented skim milk; FWH: fermented whole milk with hydrocolloids; FSH: fermented skim milk

AC C

EP

TE D

with hydrocolloids; W: whole milk, S: skim milk.

2

ACCEPTED MANUSCRIPT Table 4. Models and quality of fit for apparent viscosity at 100 s−1; G’ and G” of the three systems of model solutions. Apparent Viscosity (Pa.s) at 100 s-1 System 1

System 2

p-value

( )

OPN

-0.002

0.95

0.002

0.95

AG

0.010

0.72

0.018

0.65

GG

-0.901

0.83

-6.741

0.27

OPN * AG

-1.089

0.30

-2.363

OPN * GG

4.633

0.48

AG * GG

2.835

0.66

2

R

( )

p-value

0.004

0.81

0.011

0.54

-3.253

0.23

0.12

-1.535*

0.04

13.541

0.15

7.488*

0.09

11.199

0.22

0.996

p-value

SC

( )

( )

System 3

RI PT

Parameter

0.993

6.100

0.14

0.998

Parameter

System 1

M AN U

Storage modulus - G’ (Pa) System 2

System 3

p-value

( )

p-value

( )

OPN

1.571

0.15

1.304

0.42

1.773

0.27

AG

2.466*

0.06

1.783

0.29

3.032*

0.08

GG

-975.4*

0.01

71.92

0.76

-379.7

0.25

OPN * AG

-204.5*

0.01

-58.32

0.30

-111.2

0.13

OPN * GG

1441.9*

0.01

34.40

0.92

621.1

0.20

AG * GG

1456.1*

0,01

-16.90

0.96

576.4

0.24

R2

TE D

( )

( )

0.985

p-value

0.994

0.948

EP

Loss modulus - G” (Pa)

Parameter

( )

System 2

System 3

p-value

( )

p-value

( )

p-value

AC C

( )

System 1

OPN

0.806*

0.05

1.944*

0.10

0.483

0.25

AG

0.592*

0.10

1.009

0.33

0.724

0.12

GG

-653.5*

0.00

-167.7

0.28

384.7*

0.09

OPN * AG

-135.3*

0.00

-83.84*

0.05

43.83

0.23

OPN * GG

975.1*

0.00

342.1

0.16

-448.6

0.12

AG * GG

986.8*

0.00

313.2

0.19

-517.4*

0.10

R2

0.998

0.995

0.997

*P< 0.10. ( ) = Estimation of parameters relating to the principal effects and interactions.

3

ACCEPTED MANUSCRIPT

Table 5. Properties of the milk used to produce the FMB samples. Whole milk

Skim milk

pH

6.74 ± 0.03a

6.77 ± 0.01a

Density (g L-1)

30.80 ± 0.20a

32.58 ± 0.07b

Fat (%)

4.00 ± 0.16a

0.58 ± 0.01b

Lactose

4.78 ± 0.05a

Solids non fat (%)

8.72 ± 0.12a

Total protein (%)

3.19 ± 0.03a

RI PT

Property

4.66 ± 0.03b 8.45 ± 0.02b

SC

3.12 ± 0.02b

Data were subjected to analysis of variance and the t-test at the 5% significance level. Means in the

AC C

EP

TE D

M AN U

rows followed by the same letter are not statistically significantly different.

4

ACCEPTED MANUSCRIPT Table 6. Values of power law parameters of the FMB. Error

K (Pa.sn)

n

WH

0.9729

0.6242

40.207 ± 2.527b

0.280 ± 0.006b

SH

0.9884

3.9877

41.457 ± 2.926b

0.269 ± 0.011b

FW

0.9729

0.6242

3.199 ± 0.641a

0.315 ± 0.039c

FS

0.9673

0.5977

1.663 ± 0.240a

0.381 ± 0.004d

FWH

0.9416

5.6372

40.515 ± 7.962b

FSH

0.9581

7.0534

64.987 ± 6.401c

RI PT

R2

Sample

0.222 ± 0.038a

0.216 ± 0.017a

Data subjected to analysis of variance and the t-test at 5% significance level. Means in the columns

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followed by the same letter are not statistically significantly different.

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ACCEPTED MANUSCRIPT Table 7. Values of pH, protein content, and rheological parameters of FMB.

Sample

pH

Protein (%) c

ɳ100 (Pa.s) 1.427 ± 0.050

G’(Pa) c

242 ± 26

G”(Pa) a

87 ± 07a

WH

8.49 ± 0.06

SH

8.64 ± 0.04c

3.53 ± 0.03a

1.405 ± 0.044c

220 ± 28a

80 ± 11a

FW

4.09 ± 0.02ª

3.69 ± 0.15ab

0.151 ± 0.035a

271 ± 72b

150 ± 72b

FS

4.03 ± 0.01ª

3.93 ± 0.12b

0.096 ± 0.013a

FWH

4.80 ± 0.10b

6.19 ± 0.04c

1.098 ± 0.084b

FSH

5.22 ± 0.59b

6.53 ± 0.04c

1.731 ± 0.123d

RI PT

3.36 ± 0.02

a

299 ± 96b

84 ± 29a

299 ± 63c

140 ± 23a

569 ± 68c

221 ± 41a

Data subjected to analysis of variance and the Scott-Knott mean test at 5% significance level. Means

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in the columns followed by the same letter are not statistically significantly different.

6

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Figure 2

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Figure 3.

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(C1) Figure 4

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Figure 5.

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Figure 6.

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Figure 9.

9

ACCEPTED MANUSCRIPT Highlights

Mucilage from Peréskia aculeata Miller (OPN) was tested in hydrocolloid blends.

•

Hydrocolloid blends with OPN were applied in Fermented Milk beverages (FMB).

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Blends with OPN increased the viscosity, firmness and protein contents in FMB.

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Syneresis in FMB was reduced by hydrocolloids and was not influenced by milk fat.

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FMB with hydrocolloids blends with OPN proved to be a viable approach.

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•