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Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese emulsions
Accepted Manuscript Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese emulsions Abulimiti Kelimu, Denise Felix da Silva, Xiaolu Geng, Richard Ipsen, Anni Bygvrå Hougaard PII:
S0958-6946(17)30050-X
DOI:
10.1016/j.idairyj.2017.02.005
Reference:
INDA 4149
To appear in:
International Dairy Journal
Received Date: 21 November 2016 Revised Date:
16 February 2017
Accepted Date: 19 February 2017
Please cite this article as: Kelimu, A., Felix da Silva, D., Geng, X., Ipsen, R., Hougaard, A.B., Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese emulsions, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.02.005. 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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Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese
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emulsions
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Abulimiti Kelimua,b*, Denise Felix da Silvab, Xiaolu Gengb, Richard Ipsenb, Anni Bygvrå
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Hougaardb
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a
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830052 Urumqi, Xinjiang, China
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b
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DK-1958 Frederiksberg C, Denmark
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Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26,
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College of Food Science and pharmacy, Xinjiang Agricultural University, Nongda East Road 311,
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*Corresponding author. Tel.:
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E-mail address: [email protected] (A. Kelimu) 1
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ABSTRACT
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The influence of sodium caseinate (SC), butter milk powder (BMP) and their combinations on
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particle size, rheological properties, emulsion stability and microstructure of hot cheese emulsions
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made from mixtures of Cheddar and soft white cheese was studied. All emulsions exhibited
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shear-thinning flow behaviour and increasing SC concentration (1–4%) led to an increase in particle
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size and a decrease in apparent viscosity. In contrast, increasing BMP concentration caused
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significant decrease in particle size and slightly reduced the apparent viscosity. Stability against
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creaming and precipitation increased with increasing concentration of SC, whereas BMP
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destabilised the emulsions resulting in extensive precipitation. Confocal laser scanning microscopy
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images showed that SC exerted markedly better emulsification ability than BMP. Emulsions
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containing equal amounts of SC and BMP presented better stability against creaming and
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precipitation and this could be developed into a novel strategy to replace emulsifying salts in
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production of cheese powder.
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1.
Introduction
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Cheese powder is a convenient and ready-to-use flavouring agent made from different kinds of cheese and can be applied in wide variety of food products, such as dressings, baked foods,
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snacks, convenience foods, soups, processed cheeses and sausages (Guinee & Kilcawley, 2004;
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Varming, Andersen, Petersen, & Ardö, 2013), and even beverages.
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The procedures for cheese powder processing include cutting the cheese, adding water and emulsifying salts, mixing, emulsifying and spray drying (Písecký, 2005). For industrial scale cheese
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powder production, it is crucial to obtain a stable (i.e., without protein precipitation or creaming),
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homogenous and pumpable cheese emulsion before atomisation. Factors possibly affecting cheese
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emulsion stability preceding spray drying are type of cheese used, emulsifying salts, other optional
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ingredients, dry matter (DM) content, pH and processing conditions. Among these factors, the
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stability of cheese emulsions mostly depends on concentration and type of emulsifying salts, which
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have pH adjustment and calcium sequestering abilities, thereby improving the fat emulsification
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property of the cheese proteins (Chen & Liu, 2012; Hougaard, Sijbrandij, Varming, Ardö, & Ipsen,
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2015; Lucey, Maurer-Rothmann, & Kaliappan, 2011).
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Emulsifying salts (ES), which are mainly citrates, monophosphates, and polyphosphates,
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SC
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have been well investigated and stable cheese emulsions with good quality can be obtained in the
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presence of high (>1.5%, w/w) emulsifying salt concentrations (Hougaard et al., 2015; Kapoor &
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Metzger, 2008; Sádlíková et al., 2010; Salek et al., 2015). However, owing to the relatively high
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salt content of cheese products, addition of emulsifying salts leads to a further increase of the salt
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content of the end-product (da Cunha, Alcântara, & Viotto, 2012; Lucey et al., 2011). Therefore,
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dairy producers are under increasing pressure from health conscious consumers to reduce salt in
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their products. Furthermore, there is an increasing interest in production and application of natural
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food ingredients, which can lead to ‘clean labelling’ of the final food products.
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ACCEPTED MANUSCRIPT Sodium caseinate (SC), which consists of a mixture of the highly disordered four
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phosphoproteins: αS1-, αS2-, β- and κ-casein with hydrophilic and hydrophobic amino acids, is of
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particular interest in food emulsions because of its excellent emulsifying properties. SC has been
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widely studied for its effects on particle size, stability and rheology profiles in variety of food
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emulsions (García-Moreno, Horn, & Jacobsen, 2014; Hosseini-Parvar, Matia-Merino, & Golding,
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2015; Ihara, Ochi, Saito, & Iwatsuki, 2011; Sołowiej, Cheung, & Li-Chan, 2014). Research has also
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shown that butter milk powder (BMP), which contains phospholipids, casein, whey protein, minor
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peptides and lactose, has emulsification ability due to its protein and milk fat globule membrane
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content (Corredig & Dalgleish, 1998; Ihara et al., 2011).
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Variation in the rheological properties of an emulsion may be due to the structural changes
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caused by different emulsifying agents and the presence of SC, BMP and their combinations can
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affect cheese emulsion stability, rheological properties and microstructure. In the present study,
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which to our knowledge is the first study on the effects of adding SC and BMP on hot cheese
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emulsion stability, the individual and combined effects of the two dairy ingredients on particle size,
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rheological properties and stability was studied. Furthermore, the microstructure was examined by
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confocal laser scanning microscopy (CLSM).
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2.
Materials and methods
2.1.
Cheese types
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The cheeses used were two batches of Cheddar (Joseph Heler Cheese, Nantwich, UK) and
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one batch of soft white cheese made from ultra-filtrated milk, packaged in a rectangular aluminium
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can with brine (Bislev Dairy, Arla A/S, Nibe, Denmark).
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2.2.
Chemical analysis of the cheeses
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All chemical analyses were carried out using standard methods (AOAC, 2000): fat content
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was assessed by the Gerber-van Gulik method, total protein content was determined according to
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the Kjeldahl method by multiplying the total nitrogen with the factor 6.38 and soluble nitrogen at
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pH 4.6 was determined by acid precipitation. The pH values of the samples were measured using a
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digital pH meter (Knick pH-Meter 761 Calimatic, Berlin, Germany) at 20±2 °C. Salt was
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determined by potentiometric titration, moisture content was determined by the gravimetric method
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at 100 °C and the total content of ash was also determined gravimetrically after burning off samples
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in a muffle furnace at 525±25 °C. All chemical measurements were performed in triplicate.
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2.3.
Cheese emulsion preparation
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Cheese emulsion preparation was conducted in the dairy pilot plant of the University of
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Copenhagen. A cheese emulsion was prepared by mixing 300 g of cheddar cheese and 200 g of soft
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white cheese. From each batch of cheddar cheese and soft white cheese, seven cheese emulsions
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were produced in triplicate, leading to a total of 42 samples produced in random. The experimental
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design and the amount of relevant ingredients used are shown in Table 1. The dry matter was the
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same for all the samples (45%, w/w) as was the pH (5.7) and based on the composition of the raw
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materials calculation show that the protein content varied from 16–17.6 % and the fat content from
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22.4 to 23.5%. The influence of 4% (w/w) SC, 4% (w/w) BMP or 4% (w/w) ES on emulsion
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microstructure in comparison with a control without any additions was studied separately in
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experiments performed on similar cheeses from the same manufacturers and of same approximate
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age.
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The ingredients were prepared by adding tap water and stirring at moderate shear-rate at 5
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w/w: 90.7% protein, 0.25% lactose, 0.8% fat, 5.3% moisture, 1.5% sodium; Friesland Campina
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DMV B.V. Amersfoort, The Netherlands), BMP (composition, by w/w: 30% protein, 50% lactose,
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6% fat, 4% moisture; Fayrefield, Børkop, DK) and their mixture solutions were stored overnight at
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4 °C to attain full hydration prior to application.
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Cheeses were cut into 3×3×3 cm pieces and added into a Stephan cooker (Stephan UMC5
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electronic, Stephan u. Söhne GmbH, Hameln, Germany) together with the other ingredients. The pH
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was adjusted to 5.7±0.01 by adding 50% potassium hydroxide before mixing, except for those with
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ES (disodium hydrogen phosphate, BK Giulini, Ludwigshafen, Germany). This was followed by a
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premixing step at a speed of 1500 rpm for 5 min in the cooker and a further mixing combined with
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direct steam injection for 45 s at the same speed to get a homogenous cheese emulsion with a
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temperature of 85±3 °C. All emulsions, including the control emulsion, were homogeneous and
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finely dispersed with no visible protein aggregates or sedimentation. Dry matter measurement,
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rheological properties, particle size distribution and stability test were carried out immediately after
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preparation of the cheese emulsion samples.
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Dry matter of the cheese emulsions
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A Sartorius MA30 moisture analyser (Sartorius Weighing Technology GmBH, Göttingen,
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Germany) was used to determine the dry matter of the cheese emulsions at 75 °C by drying until
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constant weight.
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2.5.
Particle size measurement
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Particle size distributions of the emulsions were determined by Mastersizer 2000 (Malvern
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Instruments Co. Ltd., Worcestershire, UK) at room temperature (emulsion temperature 65±5 °C).
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The applied refractive index and absorption of the dispersed phase for all emulsions were set to
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1.414 and 0.001, respectively, and the refractive index employed of continuous phase (water) was
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1.330. The emulsions were diluted by adding small aliquots into a de-ionized water containing
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measurement chamber until the instrument gave an optimum obscuration rate between 15–20%.
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Particle mean diameter was expressed as volume-weighted mean diameter (D [4,3]).
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2.6.
Dynamic rheological properties
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Rheological measurements were performed by a controlled stress rheometer (AR-G2, TA
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Instruments, New Castle, DE, USA) with a rotating upper cone (bob) and fixed lower concentric
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cylinder cup measuring system (diameter 30 mm). The experiments were carried out using a 7000
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μm gap and the temperature was precisely controlled at 60 °C by a Peltier temperature control
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system. For each measurement, approximately 20 mL of hot cheese emulsion was placed into the
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interior cup. The exposed sample perimeter was covered with resin lid to minimise evaporation.
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Flow curves for each cheese emulsion were measured over 5 min with shear rate continually
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increasing from 1 to 300 s-1, followed directly by a decrease from 300 s-1 to 1 s-1 also over 5 min.
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Shear stress values of 52 points for each cheese emulsion were obtained and average shear stress
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versus shear rate for 6 trials for each formulation were used to analyse flow properties (n and κ) by
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fitting to the Power Law model (equation 1) using TA Rheology Advantage Data Analysis Software 7
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(TA Instruments) and Origin Pro9.1 (OriginLab Corporation, Northampton, MA, USA). σ = ߢγሶ
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(1)
where: ߢ is the consistency coefficient (Pa · s n) and n denotes the flow behaviour index, reflecting
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deviation from Newtonian behaviour.
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2.7.
Emulsion stability
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Evaluation of the stability of the cheese emulsion was carried out in terms of phase
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separation by centrifugation, according to the method of Hougaard et al (2015). Each 30 mL (28.7 g)
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of emulsion was transferred into a 50 mL PP graduated conical test tube with screw cap (MEUS
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R.SL., Piovedi sacco, Italy) immediately after preparation and the tube was capped to prevent
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evaporation. Subsequently, centrifugation test was performed using a SL 16R centrifuge (Thermo
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Fisher Scientific, Waltham, MA) equipped with 71 rotor and insertions for 50 mL centrifuge tubes
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with conical bottom, at intervals of 1, 2, 3, 4 and 5 min centrifugation (423 × g at 40 °C controlled
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temperature). All samples were measured in triplicate. The stability was evaluated by measuring the
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gradual changes of cream layer height on top of the emulsion, which was supposed to separate into
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three phases if the cheese emulsion was not stable. The height of middle layer was measured after 5
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min of centrifugation. Measurements were carried out manually using a ruler.
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2.8.
Confocal laser scanning microscopy
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Microsystems, Wetzlar GmbH, Wetzlar, Germany) confocal laser scanning microscope with laser
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beams Krypton/argon (488 nm) and helium/nein (543 nm). Only four types of cheese emulsions
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were observed under the CLSM, which were with or without emulsify salt, 4% BMP and 4% SC
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containing emulsions, respectively. The preparation of cheese emulsions was as described in
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previous section 2.3. The dyes used were Nile red (9-diethylamino-5H-benzoalpha-
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phenoxazine-5-one; Sigma–Aldrich, St Louis, USA) for lipids and fluorescein isothiocyanate
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isomer (FITC; Sigma–Aldrich) for proteins. The dyes were dissolved in acetone with concentration
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of 0.01% (w/v), and 0.0045% (w/v), respectively. 30 µL of each dye solution was added to the
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concave surface of a glass slide, air dried in a fume hood and wrapped with aluminium film until
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use. Cheese emulsions were examined 10±2 min after production which allowed the temperature to
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be kept between 58–65 °C. Two drops of the cheese emulsions were placed in the center of the
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concave surface of a glass slide, gently mixed with the dyes and examined under a 40×
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magnification oil immersion objective, with emission windows 500–535 nm for FITC and 580–625
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nm for Nile Red.
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2.9.
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Statistical analysis
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Statistical analyses were carried out using Origin Pro 9.1 (OriginLab Corporation,
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Northampton, MA 01060 USA) software. The Power Law model was fitted by linear curve fitting
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in Origin Pro 9.1. One-way Analysis of Variance (ANOVA) was applied to analyse the data
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statistically. Difference between mean values was determined by Pair-Sample t-Test at an α-level of 9
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5%.
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3.
Results and discussions
3.1.
Chemical composition of cheeses
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Table 2 shows the physicochemical parameters of the cheeses applied for the manufacture of
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cheese emulsions. The average values of physicochemical parameters of the two batches of Cheddar
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were similar to each other, so the results are given in average values of 6 trials.
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3.2.
Influence of the type of emulsifying ingredients on particle size
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The volume-weight mean particle size and particle size distributions of the seven cheese
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emulsions are shown in Table 3 and Fig.1. Multi-modal particle size distributions were observed for
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emulsions only containing SC or with lower ratio of BMP (samples B1S3 and B2S2), whereas
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bimodal particle size distributions were observed for the other samples. The control sample without
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emulsifying ingredients had a mean particle size around 34.4±4.6 µm with the lowest percentage
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(8.57±0.53) of small particles in the first peak. In the presence of ES, the mean particle size
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(6.7±1.2 µm) was reduced significantly (p<0.05). This reduction in particle size is the result of
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conversion of insoluble intact casein and peptide aggregates into hydrated emulsifier (El-Bakry,
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Duggan, O’Riordan, & O’Sullivan, 2010). A slight shift of the particle size distribution toward
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smaller sizes and a significant (p<0.05) decrease in the mean particle size was found for emulsions
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made using only BMP as ingredient (24.2±2.2 µm) and the combination with 3% BMP+1% SC
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(26.9±1.7 µm) compared with the control, indicating that BMP was positively correlated with
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BMP (B2S2 and B1S3) did not exhibit a significantly different (p>0.05) mean particle size
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compared with the control, though the largest mean particle size was obtained in the case of SC
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(46.3±7.3 µm), implying an inverse effect of SC on particle size reduction. In the particle size
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distributions, the appearance of a new peak/shoulder with increasing amounts of particles of size
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100–300 µm for increasing amounts of SC was seen (Fig. 1). It is difficult to compare these results
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with other studies of the effects of SC and BMP as emulsifying agents, because the cheese emulsion
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in our case is a much more complex system compared with pure oil/water emulsions that have been
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investigated extensively, and the mechanism behind these phenomena is much more complex. BMP
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contains a high amount (> 1%) of milk fat globule membrane (MFGM) material, which is rich in
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phospholipids with resulting fast emulsification and this maybe explain the observed effect (Sodini,
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Morin, Olabi, & Jiménez-Flores, 2006). In contrast, SC, exerting synergic stabilising effect via
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electrostatic repulsion and steric hindrance, induces formation of stabilised large particles by
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trapping aggregated protein particles and oil droplets in an extensively aggregated dairy protein
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containing system (Dickinson, 2013).
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3.3.
Rheological properties of cheese emulsions
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The consistency coefficient (κ) and flow behaviour index (n) values estimated from the
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Power Law model for all samples are shown in Table 4, where it is also evident that the Power Law
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model adequately fitted the experimental data, although a high chi-square (χ2) value was found for
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the ES containing emulsion. The rheological parameters of the cheese emulsions were affected by
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emulsifying agents and all samples showed non-Newtonian, shear-thinning (pseudoplastic)
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behaviour (n <1). A significantly (p<0.05) lower n and higher κ value was observed for the
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emulsion containing ES, indicating striking variation in microstructure. An increase in SC 11
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concentration led to a significant (p<0.05) increase in n and a concomitant decrease in κ, whereas
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contrary results were seen in the case of BMP. In other words, an increase in SC concentration
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caused cheese emulsions to have a more Newtonian flow behaviour, whereas the opposite tendency
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was observed when BMP concentration increased. Differences in the rheological behaviour can be assumed to be due to particle interactions
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and structural changes (Dickinson, 2000) and samples containing ES showed a marked increase in
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apparent viscosity (Fig. 2) compared with other samples, which could be associated with a
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concomitant increase in protein hydration, viscosity and consistency (Guinee & O’Kennedy, 2012).
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The rheogram of the other samples shifted downwards with maximum shift for the sample stabilised
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by SC and almost overlapped in the case of BMP and B3S1containing emulsions. The thickness of
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cheese emulsions was thus decreased by addition of these ingredients.
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The highest decrease in apparent viscosity with increasing shear rate was found for the
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sample containing SC alone and no obvious effect of BMP on emulsion viscosity was observed. We
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propose this noticeable decrease in apparent viscosity with increasing SC concentration to be
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related to the differences observed in the particle size distribution showing an increased diversity in
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particle sizes for emulsions containing SC. Bi- or multimodal particle size distributions are known
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to reduce the viscosity of dispersions, and the effect is often explained by optimised packing of
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particles of different sizes, though this might not be the entire explanation (Willenbacher &
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Georgieva, 2013). Furthermore, almost the same apparent viscosity was observed for the samples
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Control, BMP, B3S1, and likewise the samples B2S2, B1S3 had almost the same apparent viscosity.
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The Control, BMP and B3S1containing samples showed higher apparent viscosity than the B2S2
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and B1S3 containing samples (Fig. 2). These results are also in accordance with the results of the
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Power Law model parameters, particle size results and previous studies (Pal, 1996). Only a slight
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reduction in initial apparent viscosity with increasing shear rate for emulsions stabilised with B2S2
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and B1S3 was observed, which, we hypothesise, is indicative of a more stable structure formation
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with good emulsification.
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3.4.
Influence of the type of ingredients on cheese emulsion stability
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For all cheese emulsion samples, there was cream layer formation irrespective of the type of
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ingredients added, and except for ES and B2S2containing emulsions, phase separation was
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observed for others after 5 min of centrifugation (Fig.3). Creaming might be the result of the coarse
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emulsifying nature of processing in the Stephan cooker, which led to formation of larger fat droplets
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in the cheese emulsions and resulted in creaming (Álvarez Cerimedo, Iriart, Candal, & Herrera,
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2010).
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Samples stabilised by ES or a 50:50 mixtures of SC and BMP (B2S2) showed good stability without visible sedimentation, but protein precipitation was seen for the remaining samples after
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centrifugation (Table 6). The observed improvement in stability of cheese emulsion by B2S2
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addition might be the result of synergic effect of BMP and SC, where the MFGM (phospholipids)
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content in BMP ensured fat emulsification, while SC exerted emulsification and restructuring
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effects by steric and electrostatic interactions (Horne, 2008). Although the highest degree of creaming was observed, cheese emulsions without
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emulsifying agents exhibited some degree of stability against precipitation, indicating slight effects
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of inherent proteins and peptides for stable structure formation. (Ray, Gholamhosseinpour, Ipsen, &
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Hougaard, 2016). This could also be explained by the fact that the adjustment of pH increased the
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extent of native intact casein hydration, whereby the emulsion structure is stabilised (Lu, Shirashoji,
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& Lucey, 2008).
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Similar extent of creaming and significant (p<0.05) sedimentation was observed for cheese
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emulsions stabilised with BMP, which may be the result of bridging flocculation and coalescence,
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owing, most likely, to the lack of enough soluble protein to form a homogenous structure. Generally, 13
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stability of emulsions is related to particle size, which appears to not be the case for cheese
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emulsions, where the formation of a stable structure by protein interaction is also crucial for
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keeping the stability of an emulsion. In the presence of SC alone, although exhibiting some degree of creaming, stability against
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precipitation was significantly (p<0.05) improved and remarkable impact against creaming and
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precipitation was observed, which is in accordance with other reports (Liang et al., 2014; Ye, 2008).
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As for the different combinations of BMP and SC, no obvious difference in creaming was
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observed, whereas B1S3 has significant (p<0.05) effect against precipitation. Moreover, the B2S2
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combination showed good stability with no observable protein sedimentation, indicating that the
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inclusion of this combination is optimum for the stability of the present type of cheese emulsion.
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3.5.
Microstructure of cheese emulsion evaluated by CLSM
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To further confirm the effects of SC, BMP and ES on the rheological properties and particle
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size variations, the microstructure of cheese emulsion samples was examined by CLSM (Fig. 4). As
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expected, different emulsifying ingredients affected the cheese emulsion morphology considerably.
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In the absence of emulsifying ingredients, the CLSM image revealed a microstructure of discrete,
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coarse, irregular (non-spherical) protein aggregates (green), void area (black) and fat clusters (red),
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in which protein and fat droplets were disconnected from each other and with little or no indication
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of emulsification (Fig. 4B). The cheese emulsion containing ES showed the presence of relatively
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evenly dispersed small fat droplets that were entrapped within a homogeneous protein structure,
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clearly indicating the great impact of ES on protein emulsification (Fig. 4A). In the case of a BMP
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containing emulsion, the confocal images exhibited a reduction in particle size and relatively evenly
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distributed and smaller fat droplets compared with the control (Fig. 4C). The reason for the effects
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of BMP on cheese emulsion structure may be the result of slight emulsifying effect of milk protein
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(whey protein and casein) and/or MFGM in BMP, the main reason is not presently known, but will
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be the subject of future studies. In contrast to the control sample, the image of the SC containing
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emulsion exhibited bigger protein particles, the surface of which was coated by fat droplets in a thin
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layer (yellowish), indicating an improvement of the emulsion stability against creaming (Fig. 4D). These observations could aid in explaining the differences observed, especially in the
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particle size distributions, where the increase in particle size and shoulder formation in the size
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distributions could be suggested to be due to increased interactions between particles of protein and
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fat droplets caused by the emulsifying properties of the SC. The marked differences observed
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between the particle size distributions for samples with ES and the remaining samples could most
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likely be due to the fact that the ES addition causes formation of a continuous protein network in the
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samples.
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SC led to the lowest level of creaming after the ES and this could also be related to the
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increased level of interactions observed (Fig. 4D) between SC and fat droplets. Again the observed
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effects are difficult to compare with other studies of SC as emulsifier, because of the complex
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nature of the cheese emulsion. However, it could be suggested that the cheese protein particles are
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more or less inactive in the emulsification and the effects of SC are similar to those observed by
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Liang et al. (2014) and Ye et al. ( 2008) where creaming and emulsion viscosity is seen to be
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affected by SC in a concentration dependent manner also influenced by other constituents of the
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emulsions.
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Conclusion
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Dairy derived ingredients affected the stability, rheological properties and microstructure of
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hot cheese emulsion for cheese powder manufacture. Increasing SC concentration led to larger
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particles, decrease in viscosity, improved fat emulsification as well as better emulsion stability. On 15
ACCEPTED MANUSCRIPT the contrary, due to the lack of stable structure formation, a cheese emulsion containing BMP solely,
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although it exhibited decrease in particle size, showed instability by creaming and precipitation.
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However, different combinations (B1S3, B2S2, B3S1) of SC and BMP improved the overall
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stability of cheese emulsion against creaming and precipitation. A desirable cheese emulsion
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stability was obtained by addition of equal amounts of the two ingredients (the B2S2 combination).
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This might be mainly attributed to the dual effects of SC as restructuring and emulsifying, which is
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likely to improve in the presence of BMP. Based on the results presented so far, it could be
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suggested that, to some extent, particle size does not affect stability of cheese emulsion significantly,
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where fat emulsification and protein stabilization play equally important roles in keeping cheese
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emulsion stability. Overall, the influence of SC and BMP on rheological properties and stability of
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hot cheese emulsion may be of practical importance and has potential application as ES replacers.
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Acknowledgements
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The authors thank the China Scholarship Council (CSC) and Lactosan A/S for providing financial support for this research.
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Sołowiej, B., Cheung, I. W. Y., & Li-Chan, E. C. Y. (2014). Texture, rheology and meltability of processed cheese analogues prepared using rennet or acid casein with or without added
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Varming, C., Andersen, L. T., Petersen, M. A., & Ardö, Y. (2013). Flavour compounds and sensory
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49). Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.
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Ye, A. (2008). Interfacial composition and stability of emulsions made with mixtures of commercial sodium caseinate and whey protein concentrate. Food Chemistry, 110, 946–952.
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Figure legends
2
Fig. 1. Particle size distribution of seven cheese emulsion samples containing different ingredients
4
(■, control; ●, emulsifying salt; □, sodium caseinate; ○, butter milk powder; ▽, 1% butter milk
5
powder + 3% sodium caseinate; ★, 2% butter milk powder + 2% sodium caseinate; ▼, 3% butter
6
milk powder + 1% sodium caseinate).
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Fig. 2. Apparent viscosity variation of cheese emulsions as a function of shear rate (□, control; ●,
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emulsifying salt; ▲, sodium caseinate; 〇, butter milk powder; ▽, 1% butter milk powder + 3%
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sodium caseinate; △, 2% butter milk powder + 2% sodium caseinate; +, 3% butter milk powder +
11
1% sodium caseinate).
12
Fig. 3. Creaming of different samples under centrifugation (□, control; ●, emulsifying salt; ▲,
14
sodium caseinate; ■, butter milk powder; △, 1% butter milk powder + 3% sodium caseinate; ▽,
15
2% butter milk powder + 2% sodium caseinate; ○, 3% butter milk powder +1% sodium caseinate).
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Fig. 4. Confocal laser scanning microscopy images of cheese feeds: A, cheese feed containing
18
emulsifying salt; B, cheese feed without any additions; C, cheese feed with 4% w/w (on dry matter
19
basis) added buttermilk powder; D, cheese feed with 4% w/w (on dry matter basis) added sodium
20
caseinate. Representative images are chosen from a series of images available for each sample
21
composition.
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1
ACCEPTED MANUSCRIPT Table 1
Control ES BMP SC B1S3 B2S2 B3S1
Abbreviations are: ES, emulsifying salt; BMP, butter milk powder; SC, sodium caseinate; B1S3, 1%
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Ingredients (w/w, by dry matter) ES BMP SC Water (g) 117 4% 136 4% 136 4% 136 1% 3% 136 2% 2% 136 3% 1% 136
SC
Sample
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Experimental design and ingredients used.a
butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate;
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B3S1, 1% butter milk powder+3% sodium caseinate.
ACCEPTED MANUSCRIPT Table 2 Physicochemical parameters of the two different types of cheeses. Ash (%)
pH
1.67±0.06 4.48±0.02
3.95±0.02 5.77±0.02
5.46±0.01 4.60±0.00
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pH 4.6 Soluble N (%) 0.66±0.02 0.37±0.01
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Salt (%)
SC
Protein Fat (%) (%) 24.38±0.15 36.8±0.00 15.26±0.18 17.17±0.29
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Cheddar Soft white
Moisture (%) 34.28±0.12 60.37±0.13
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Cheese
ACCEPTED MANUSCRIPT Table 3
Control
34.4 ± 4.6a
ES
6.7 ± 1.2c
SC
46.3 ± 7.3a
BMP
24.2 ± 2.2b
B1S3
44.2 ± 9.0a
B2S2
38.4 ± 4.2a
B3S1
26.9 ± 1.7b
SC
D [4, 3] (µm)
Abbreviations are: ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3,1%
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Sample
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Average particle sizes of cheese emulsions. a
butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 3% butter milk powder + 1% sodium caseinate. Values are means ± SD (n = 6); means with
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different superscript letters are significantly different (p<0.05).
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κ (Pa sn) 0.07 ± 0.02c 2.93 ± 0.42a 0.03 ± 0.02d 0.13 ± 0.04b 0.04 ± 0.02d 0.07 ± 0.06b,c 0.10 ± 0.03b,c
a
n 0.87 ± 0.02b 0.64 ± 0.03d 0.95 ± 0.06a 0.77 ± 0.04c 0.90 ± 0.07a,b 0.88 ± 0.10a,b 0.79 ± 0.03c
R2 0.979 0.967 0.988 0.968 0.978 0.966 0.964
χ2 0.197 26.59 0.014 0.190 0.057 0.155 0.223
SC
Sample Control ES SC BMP B1S3 B2S2 B3S1
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Power Law model parameters.a
Abbreviations are: κ, consistency coefficient; n, flow behaviour index; R2, determination
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coefficient; χ2, chi-square; ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3, 1% butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 1% butter milk powder + 3% sodium caseinate. Values are means ± SD (n=6); means in the same column followed by different superscript letters are significantly different
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(p<0.05).
ACCEPTED MANUSCRIPT Table 5 Height of middle phase after centrifugation. a Height (mm)
ES
No observable boundary
Control
25.3 ± 0.6b
SC
18.6 ± 1.1c
BMP
29.7 ± 1.2a
B1S3
19.8 ± 1.8c
B2S2
No observable boundary
B3S1
22.9 ± 1.1b
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Formulation
Abbreviations are: ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3,
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1% butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 1% butter milk powder + 3% sodium caseinate. Values are means ± SD (n=6);
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means followed by different superscript letters are significantly different (p<0.05).
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Particle volume (%)
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Fig 1
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Particle diameter (µm)
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Fig. 2
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Apparent viscosity (Pa s) (%)
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Shear rate (1/s)
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Fig. 3
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Height of fat layer (mm)
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Time (min)
Fig. 4
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S0958-6946(17)30050-X
DOI:
10.1016/j.idairyj.2017.02.005
Reference:
INDA 4149
To appear in:
International Dairy Journal
Received Date: 21 November 2016 Revised Date:
16 February 2017
Accepted Date: 19 February 2017
Please cite this article as: Kelimu, A., Felix da Silva, D., Geng, X., Ipsen, R., Hougaard, A.B., Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese emulsions, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.02.005. 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.
ACCEPTED MANUSCRIPT 1
Effects of different dairy ingredients on the rheological behaviour and stability of hot cheese
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emulsions
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Abulimiti Kelimua,b*, Denise Felix da Silvab, Xiaolu Gengb, Richard Ipsenb, Anni Bygvrå
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Hougaardb
SC
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a
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830052 Urumqi, Xinjiang, China
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b
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DK-1958 Frederiksberg C, Denmark
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Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26,
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College of Food Science and pharmacy, Xinjiang Agricultural University, Nongda East Road 311,
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*Corresponding author. Tel.:
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E-mail address: [email protected] (A. Kelimu) 1
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________________________________________________________________________________
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ABSTRACT
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The influence of sodium caseinate (SC), butter milk powder (BMP) and their combinations on
30
particle size, rheological properties, emulsion stability and microstructure of hot cheese emulsions
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made from mixtures of Cheddar and soft white cheese was studied. All emulsions exhibited
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shear-thinning flow behaviour and increasing SC concentration (1–4%) led to an increase in particle
33
size and a decrease in apparent viscosity. In contrast, increasing BMP concentration caused
34
significant decrease in particle size and slightly reduced the apparent viscosity. Stability against
35
creaming and precipitation increased with increasing concentration of SC, whereas BMP
36
destabilised the emulsions resulting in extensive precipitation. Confocal laser scanning microscopy
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images showed that SC exerted markedly better emulsification ability than BMP. Emulsions
38
containing equal amounts of SC and BMP presented better stability against creaming and
39
precipitation and this could be developed into a novel strategy to replace emulsifying salts in
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production of cheese powder.
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________________________________________________________________________________
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1.
Introduction
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Cheese powder is a convenient and ready-to-use flavouring agent made from different kinds of cheese and can be applied in wide variety of food products, such as dressings, baked foods,
48
snacks, convenience foods, soups, processed cheeses and sausages (Guinee & Kilcawley, 2004;
49
Varming, Andersen, Petersen, & Ardö, 2013), and even beverages.
50
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The procedures for cheese powder processing include cutting the cheese, adding water and emulsifying salts, mixing, emulsifying and spray drying (Písecký, 2005). For industrial scale cheese
52
powder production, it is crucial to obtain a stable (i.e., without protein precipitation or creaming),
53
homogenous and pumpable cheese emulsion before atomisation. Factors possibly affecting cheese
54
emulsion stability preceding spray drying are type of cheese used, emulsifying salts, other optional
55
ingredients, dry matter (DM) content, pH and processing conditions. Among these factors, the
56
stability of cheese emulsions mostly depends on concentration and type of emulsifying salts, which
57
have pH adjustment and calcium sequestering abilities, thereby improving the fat emulsification
58
property of the cheese proteins (Chen & Liu, 2012; Hougaard, Sijbrandij, Varming, Ardö, & Ipsen,
59
2015; Lucey, Maurer-Rothmann, & Kaliappan, 2011).
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Emulsifying salts (ES), which are mainly citrates, monophosphates, and polyphosphates,
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SC
51
have been well investigated and stable cheese emulsions with good quality can be obtained in the
62
presence of high (>1.5%, w/w) emulsifying salt concentrations (Hougaard et al., 2015; Kapoor &
63
Metzger, 2008; Sádlíková et al., 2010; Salek et al., 2015). However, owing to the relatively high
64
salt content of cheese products, addition of emulsifying salts leads to a further increase of the salt
65
content of the end-product (da Cunha, Alcântara, & Viotto, 2012; Lucey et al., 2011). Therefore,
66
dairy producers are under increasing pressure from health conscious consumers to reduce salt in
67
their products. Furthermore, there is an increasing interest in production and application of natural
68
food ingredients, which can lead to ‘clean labelling’ of the final food products.
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3
ACCEPTED MANUSCRIPT Sodium caseinate (SC), which consists of a mixture of the highly disordered four
70
phosphoproteins: αS1-, αS2-, β- and κ-casein with hydrophilic and hydrophobic amino acids, is of
71
particular interest in food emulsions because of its excellent emulsifying properties. SC has been
72
widely studied for its effects on particle size, stability and rheology profiles in variety of food
73
emulsions (García-Moreno, Horn, & Jacobsen, 2014; Hosseini-Parvar, Matia-Merino, & Golding,
74
2015; Ihara, Ochi, Saito, & Iwatsuki, 2011; Sołowiej, Cheung, & Li-Chan, 2014). Research has also
75
shown that butter milk powder (BMP), which contains phospholipids, casein, whey protein, minor
76
peptides and lactose, has emulsification ability due to its protein and milk fat globule membrane
77
content (Corredig & Dalgleish, 1998; Ihara et al., 2011).
SC
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Variation in the rheological properties of an emulsion may be due to the structural changes
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caused by different emulsifying agents and the presence of SC, BMP and their combinations can
80
affect cheese emulsion stability, rheological properties and microstructure. In the present study,
81
which to our knowledge is the first study on the effects of adding SC and BMP on hot cheese
82
emulsion stability, the individual and combined effects of the two dairy ingredients on particle size,
83
rheological properties and stability was studied. Furthermore, the microstructure was examined by
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confocal laser scanning microscopy (CLSM).
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2.
Materials and methods
2.1.
Cheese types
87 88 89 90
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The cheeses used were two batches of Cheddar (Joseph Heler Cheese, Nantwich, UK) and
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one batch of soft white cheese made from ultra-filtrated milk, packaged in a rectangular aluminium
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can with brine (Bislev Dairy, Arla A/S, Nibe, Denmark).
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2.2.
Chemical analysis of the cheeses
95
All chemical analyses were carried out using standard methods (AOAC, 2000): fat content
96
was assessed by the Gerber-van Gulik method, total protein content was determined according to
98
the Kjeldahl method by multiplying the total nitrogen with the factor 6.38 and soluble nitrogen at
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pH 4.6 was determined by acid precipitation. The pH values of the samples were measured using a
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digital pH meter (Knick pH-Meter 761 Calimatic, Berlin, Germany) at 20±2 °C. Salt was
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determined by potentiometric titration, moisture content was determined by the gravimetric method
102
at 100 °C and the total content of ash was also determined gravimetrically after burning off samples
103
in a muffle furnace at 525±25 °C. All chemical measurements were performed in triplicate.
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2.3.
Cheese emulsion preparation
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Cheese emulsion preparation was conducted in the dairy pilot plant of the University of
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Copenhagen. A cheese emulsion was prepared by mixing 300 g of cheddar cheese and 200 g of soft
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white cheese. From each batch of cheddar cheese and soft white cheese, seven cheese emulsions
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were produced in triplicate, leading to a total of 42 samples produced in random. The experimental
111
design and the amount of relevant ingredients used are shown in Table 1. The dry matter was the
112
same for all the samples (45%, w/w) as was the pH (5.7) and based on the composition of the raw
113
materials calculation show that the protein content varied from 16–17.6 % and the fat content from
114
22.4 to 23.5%. The influence of 4% (w/w) SC, 4% (w/w) BMP or 4% (w/w) ES on emulsion
115
microstructure in comparison with a control without any additions was studied separately in
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experiments performed on similar cheeses from the same manufacturers and of same approximate
117
age.
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The ingredients were prepared by adding tap water and stirring at moderate shear-rate at 5
ACCEPTED MANUSCRIPT 30 °C on a magnetic stirrer until the ingredients were completely dissolved. SC (composition by
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w/w: 90.7% protein, 0.25% lactose, 0.8% fat, 5.3% moisture, 1.5% sodium; Friesland Campina
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DMV B.V. Amersfoort, The Netherlands), BMP (composition, by w/w: 30% protein, 50% lactose,
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6% fat, 4% moisture; Fayrefield, Børkop, DK) and their mixture solutions were stored overnight at
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4 °C to attain full hydration prior to application.
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Cheeses were cut into 3×3×3 cm pieces and added into a Stephan cooker (Stephan UMC5
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electronic, Stephan u. Söhne GmbH, Hameln, Germany) together with the other ingredients. The pH
126
was adjusted to 5.7±0.01 by adding 50% potassium hydroxide before mixing, except for those with
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ES (disodium hydrogen phosphate, BK Giulini, Ludwigshafen, Germany). This was followed by a
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premixing step at a speed of 1500 rpm for 5 min in the cooker and a further mixing combined with
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direct steam injection for 45 s at the same speed to get a homogenous cheese emulsion with a
130
temperature of 85±3 °C. All emulsions, including the control emulsion, were homogeneous and
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finely dispersed with no visible protein aggregates or sedimentation. Dry matter measurement,
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rheological properties, particle size distribution and stability test were carried out immediately after
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preparation of the cheese emulsion samples.
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2.4.
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Dry matter of the cheese emulsions
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A Sartorius MA30 moisture analyser (Sartorius Weighing Technology GmBH, Göttingen,
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Germany) was used to determine the dry matter of the cheese emulsions at 75 °C by drying until
139
constant weight.
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2.5.
Particle size measurement
6
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Particle size distributions of the emulsions were determined by Mastersizer 2000 (Malvern
143
Instruments Co. Ltd., Worcestershire, UK) at room temperature (emulsion temperature 65±5 °C).
145
The applied refractive index and absorption of the dispersed phase for all emulsions were set to
146
1.414 and 0.001, respectively, and the refractive index employed of continuous phase (water) was
147
1.330. The emulsions were diluted by adding small aliquots into a de-ionized water containing
148
measurement chamber until the instrument gave an optimum obscuration rate between 15–20%.
149
Particle mean diameter was expressed as volume-weighted mean diameter (D [4,3]).
150 151
2.6.
Dynamic rheological properties
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Rheological measurements were performed by a controlled stress rheometer (AR-G2, TA
154
Instruments, New Castle, DE, USA) with a rotating upper cone (bob) and fixed lower concentric
155
cylinder cup measuring system (diameter 30 mm). The experiments were carried out using a 7000
156
μm gap and the temperature was precisely controlled at 60 °C by a Peltier temperature control
157
system. For each measurement, approximately 20 mL of hot cheese emulsion was placed into the
158
interior cup. The exposed sample perimeter was covered with resin lid to minimise evaporation.
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Flow curves for each cheese emulsion were measured over 5 min with shear rate continually
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increasing from 1 to 300 s-1, followed directly by a decrease from 300 s-1 to 1 s-1 also over 5 min.
161
Shear stress values of 52 points for each cheese emulsion were obtained and average shear stress
162
versus shear rate for 6 trials for each formulation were used to analyse flow properties (n and κ) by
163
fitting to the Power Law model (equation 1) using TA Rheology Advantage Data Analysis Software 7
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(TA Instruments) and Origin Pro9.1 (OriginLab Corporation, Northampton, MA, USA). σ = ߢγሶ
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(1)
where: ߢ is the consistency coefficient (Pa · s n) and n denotes the flow behaviour index, reflecting
167
deviation from Newtonian behaviour.
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2.7.
Emulsion stability
SC
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Evaluation of the stability of the cheese emulsion was carried out in terms of phase
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separation by centrifugation, according to the method of Hougaard et al (2015). Each 30 mL (28.7 g)
173
of emulsion was transferred into a 50 mL PP graduated conical test tube with screw cap (MEUS
174
R.SL., Piovedi sacco, Italy) immediately after preparation and the tube was capped to prevent
175
evaporation. Subsequently, centrifugation test was performed using a SL 16R centrifuge (Thermo
176
Fisher Scientific, Waltham, MA) equipped with 71 rotor and insertions for 50 mL centrifuge tubes
177
with conical bottom, at intervals of 1, 2, 3, 4 and 5 min centrifugation (423 × g at 40 °C controlled
178
temperature). All samples were measured in triplicate. The stability was evaluated by measuring the
179
gradual changes of cream layer height on top of the emulsion, which was supposed to separate into
180
three phases if the cheese emulsion was not stable. The height of middle layer was measured after 5
181
min of centrifugation. Measurements were carried out manually using a ruler.
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182 183
2.8.
Confocal laser scanning microscopy
184
8
ACCEPTED MANUSCRIPT Visualisation of the cheese emulsions was carried out by using a Leica SP5 (Leica
186
Microsystems, Wetzlar GmbH, Wetzlar, Germany) confocal laser scanning microscope with laser
187
beams Krypton/argon (488 nm) and helium/nein (543 nm). Only four types of cheese emulsions
188
were observed under the CLSM, which were with or without emulsify salt, 4% BMP and 4% SC
189
containing emulsions, respectively. The preparation of cheese emulsions was as described in
190
previous section 2.3. The dyes used were Nile red (9-diethylamino-5H-benzoalpha-
191
phenoxazine-5-one; Sigma–Aldrich, St Louis, USA) for lipids and fluorescein isothiocyanate
192
isomer (FITC; Sigma–Aldrich) for proteins. The dyes were dissolved in acetone with concentration
193
of 0.01% (w/v), and 0.0045% (w/v), respectively. 30 µL of each dye solution was added to the
194
concave surface of a glass slide, air dried in a fume hood and wrapped with aluminium film until
195
use. Cheese emulsions were examined 10±2 min after production which allowed the temperature to
196
be kept between 58–65 °C. Two drops of the cheese emulsions were placed in the center of the
197
concave surface of a glass slide, gently mixed with the dyes and examined under a 40×
198
magnification oil immersion objective, with emission windows 500–535 nm for FITC and 580–625
199
nm for Nile Red.
201 202
2.9.
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Statistical analysis
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Statistical analyses were carried out using Origin Pro 9.1 (OriginLab Corporation,
204
Northampton, MA 01060 USA) software. The Power Law model was fitted by linear curve fitting
205
in Origin Pro 9.1. One-way Analysis of Variance (ANOVA) was applied to analyse the data
206
statistically. Difference between mean values was determined by Pair-Sample t-Test at an α-level of 9
ACCEPTED MANUSCRIPT 207
5%.
208 209
3.
Results and discussions
3.1.
Chemical composition of cheeses
211
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212
Table 2 shows the physicochemical parameters of the cheeses applied for the manufacture of
214
cheese emulsions. The average values of physicochemical parameters of the two batches of Cheddar
215
were similar to each other, so the results are given in average values of 6 trials.
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216 217
3.2.
Influence of the type of emulsifying ingredients on particle size
218
The volume-weight mean particle size and particle size distributions of the seven cheese
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emulsions are shown in Table 3 and Fig.1. Multi-modal particle size distributions were observed for
221
emulsions only containing SC or with lower ratio of BMP (samples B1S3 and B2S2), whereas
222
bimodal particle size distributions were observed for the other samples. The control sample without
223
emulsifying ingredients had a mean particle size around 34.4±4.6 µm with the lowest percentage
224
(8.57±0.53) of small particles in the first peak. In the presence of ES, the mean particle size
225
(6.7±1.2 µm) was reduced significantly (p<0.05). This reduction in particle size is the result of
226
conversion of insoluble intact casein and peptide aggregates into hydrated emulsifier (El-Bakry,
227
Duggan, O’Riordan, & O’Sullivan, 2010). A slight shift of the particle size distribution toward
228
smaller sizes and a significant (p<0.05) decrease in the mean particle size was found for emulsions
229
made using only BMP as ingredient (24.2±2.2 µm) and the combination with 3% BMP+1% SC
230
(26.9±1.7 µm) compared with the control, indicating that BMP was positively correlated with
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ACCEPTED MANUSCRIPT particle size reduction. However, emulsions stabilised with only SC or mixtures containing less
232
BMP (B2S2 and B1S3) did not exhibit a significantly different (p>0.05) mean particle size
233
compared with the control, though the largest mean particle size was obtained in the case of SC
234
(46.3±7.3 µm), implying an inverse effect of SC on particle size reduction. In the particle size
235
distributions, the appearance of a new peak/shoulder with increasing amounts of particles of size
236
100–300 µm for increasing amounts of SC was seen (Fig. 1). It is difficult to compare these results
237
with other studies of the effects of SC and BMP as emulsifying agents, because the cheese emulsion
238
in our case is a much more complex system compared with pure oil/water emulsions that have been
239
investigated extensively, and the mechanism behind these phenomena is much more complex. BMP
240
contains a high amount (> 1%) of milk fat globule membrane (MFGM) material, which is rich in
241
phospholipids with resulting fast emulsification and this maybe explain the observed effect (Sodini,
242
Morin, Olabi, & Jiménez-Flores, 2006). In contrast, SC, exerting synergic stabilising effect via
243
electrostatic repulsion and steric hindrance, induces formation of stabilised large particles by
244
trapping aggregated protein particles and oil droplets in an extensively aggregated dairy protein
245
containing system (Dickinson, 2013).
248 249
3.3.
Rheological properties of cheese emulsions
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The consistency coefficient (κ) and flow behaviour index (n) values estimated from the
250
Power Law model for all samples are shown in Table 4, where it is also evident that the Power Law
251
model adequately fitted the experimental data, although a high chi-square (χ2) value was found for
252
the ES containing emulsion. The rheological parameters of the cheese emulsions were affected by
253
emulsifying agents and all samples showed non-Newtonian, shear-thinning (pseudoplastic)
254
behaviour (n <1). A significantly (p<0.05) lower n and higher κ value was observed for the
255
emulsion containing ES, indicating striking variation in microstructure. An increase in SC 11
ACCEPTED MANUSCRIPT 256
concentration led to a significant (p<0.05) increase in n and a concomitant decrease in κ, whereas
257
contrary results were seen in the case of BMP. In other words, an increase in SC concentration
258
caused cheese emulsions to have a more Newtonian flow behaviour, whereas the opposite tendency
259
was observed when BMP concentration increased. Differences in the rheological behaviour can be assumed to be due to particle interactions
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and structural changes (Dickinson, 2000) and samples containing ES showed a marked increase in
262
apparent viscosity (Fig. 2) compared with other samples, which could be associated with a
263
concomitant increase in protein hydration, viscosity and consistency (Guinee & O’Kennedy, 2012).
264
The rheogram of the other samples shifted downwards with maximum shift for the sample stabilised
265
by SC and almost overlapped in the case of BMP and B3S1containing emulsions. The thickness of
266
cheese emulsions was thus decreased by addition of these ingredients.
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The highest decrease in apparent viscosity with increasing shear rate was found for the
268
sample containing SC alone and no obvious effect of BMP on emulsion viscosity was observed. We
269
propose this noticeable decrease in apparent viscosity with increasing SC concentration to be
270
related to the differences observed in the particle size distribution showing an increased diversity in
271
particle sizes for emulsions containing SC. Bi- or multimodal particle size distributions are known
272
to reduce the viscosity of dispersions, and the effect is often explained by optimised packing of
273
particles of different sizes, though this might not be the entire explanation (Willenbacher &
274
Georgieva, 2013). Furthermore, almost the same apparent viscosity was observed for the samples
275
Control, BMP, B3S1, and likewise the samples B2S2, B1S3 had almost the same apparent viscosity.
276
The Control, BMP and B3S1containing samples showed higher apparent viscosity than the B2S2
277
and B1S3 containing samples (Fig. 2). These results are also in accordance with the results of the
278
Power Law model parameters, particle size results and previous studies (Pal, 1996). Only a slight
279
reduction in initial apparent viscosity with increasing shear rate for emulsions stabilised with B2S2
280
and B1S3 was observed, which, we hypothesise, is indicative of a more stable structure formation
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12
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with good emulsification.
282 283
3.4.
Influence of the type of ingredients on cheese emulsion stability
284
For all cheese emulsion samples, there was cream layer formation irrespective of the type of
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ingredients added, and except for ES and B2S2containing emulsions, phase separation was
287
observed for others after 5 min of centrifugation (Fig.3). Creaming might be the result of the coarse
288
emulsifying nature of processing in the Stephan cooker, which led to formation of larger fat droplets
289
in the cheese emulsions and resulted in creaming (Álvarez Cerimedo, Iriart, Candal, & Herrera,
290
2010).
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SC
286
Samples stabilised by ES or a 50:50 mixtures of SC and BMP (B2S2) showed good stability without visible sedimentation, but protein precipitation was seen for the remaining samples after
293
centrifugation (Table 6). The observed improvement in stability of cheese emulsion by B2S2
294
addition might be the result of synergic effect of BMP and SC, where the MFGM (phospholipids)
295
content in BMP ensured fat emulsification, while SC exerted emulsification and restructuring
296
effects by steric and electrostatic interactions (Horne, 2008). Although the highest degree of creaming was observed, cheese emulsions without
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emulsifying agents exhibited some degree of stability against precipitation, indicating slight effects
299
of inherent proteins and peptides for stable structure formation. (Ray, Gholamhosseinpour, Ipsen, &
300
Hougaard, 2016). This could also be explained by the fact that the adjustment of pH increased the
301
extent of native intact casein hydration, whereby the emulsion structure is stabilised (Lu, Shirashoji,
302
& Lucey, 2008).
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303
Similar extent of creaming and significant (p<0.05) sedimentation was observed for cheese
304
emulsions stabilised with BMP, which may be the result of bridging flocculation and coalescence,
305
owing, most likely, to the lack of enough soluble protein to form a homogenous structure. Generally, 13
ACCEPTED MANUSCRIPT 306
stability of emulsions is related to particle size, which appears to not be the case for cheese
307
emulsions, where the formation of a stable structure by protein interaction is also crucial for
308
keeping the stability of an emulsion. In the presence of SC alone, although exhibiting some degree of creaming, stability against
309
precipitation was significantly (p<0.05) improved and remarkable impact against creaming and
311
precipitation was observed, which is in accordance with other reports (Liang et al., 2014; Ye, 2008).
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As for the different combinations of BMP and SC, no obvious difference in creaming was
312
observed, whereas B1S3 has significant (p<0.05) effect against precipitation. Moreover, the B2S2
314
combination showed good stability with no observable protein sedimentation, indicating that the
315
inclusion of this combination is optimum for the stability of the present type of cheese emulsion.
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313
316 317
3.5.
Microstructure of cheese emulsion evaluated by CLSM
318
To further confirm the effects of SC, BMP and ES on the rheological properties and particle
320
size variations, the microstructure of cheese emulsion samples was examined by CLSM (Fig. 4). As
321
expected, different emulsifying ingredients affected the cheese emulsion morphology considerably.
322
In the absence of emulsifying ingredients, the CLSM image revealed a microstructure of discrete,
323
coarse, irregular (non-spherical) protein aggregates (green), void area (black) and fat clusters (red),
324
in which protein and fat droplets were disconnected from each other and with little or no indication
325
of emulsification (Fig. 4B). The cheese emulsion containing ES showed the presence of relatively
326
evenly dispersed small fat droplets that were entrapped within a homogeneous protein structure,
327
clearly indicating the great impact of ES on protein emulsification (Fig. 4A). In the case of a BMP
328
containing emulsion, the confocal images exhibited a reduction in particle size and relatively evenly
329
distributed and smaller fat droplets compared with the control (Fig. 4C). The reason for the effects
330
of BMP on cheese emulsion structure may be the result of slight emulsifying effect of milk protein
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14
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(whey protein and casein) and/or MFGM in BMP, the main reason is not presently known, but will
332
be the subject of future studies. In contrast to the control sample, the image of the SC containing
333
emulsion exhibited bigger protein particles, the surface of which was coated by fat droplets in a thin
334
layer (yellowish), indicating an improvement of the emulsion stability against creaming (Fig. 4D). These observations could aid in explaining the differences observed, especially in the
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particle size distributions, where the increase in particle size and shoulder formation in the size
337
distributions could be suggested to be due to increased interactions between particles of protein and
338
fat droplets caused by the emulsifying properties of the SC. The marked differences observed
339
between the particle size distributions for samples with ES and the remaining samples could most
340
likely be due to the fact that the ES addition causes formation of a continuous protein network in the
341
samples.
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SC led to the lowest level of creaming after the ES and this could also be related to the
342
increased level of interactions observed (Fig. 4D) between SC and fat droplets. Again the observed
344
effects are difficult to compare with other studies of SC as emulsifier, because of the complex
345
nature of the cheese emulsion. However, it could be suggested that the cheese protein particles are
346
more or less inactive in the emulsification and the effects of SC are similar to those observed by
347
Liang et al. (2014) and Ye et al. ( 2008) where creaming and emulsion viscosity is seen to be
348
affected by SC in a concentration dependent manner also influenced by other constituents of the
349
emulsions.
350 351
4.
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Conclusion
352 353
Dairy derived ingredients affected the stability, rheological properties and microstructure of
354
hot cheese emulsion for cheese powder manufacture. Increasing SC concentration led to larger
355
particles, decrease in viscosity, improved fat emulsification as well as better emulsion stability. On 15
ACCEPTED MANUSCRIPT the contrary, due to the lack of stable structure formation, a cheese emulsion containing BMP solely,
357
although it exhibited decrease in particle size, showed instability by creaming and precipitation.
358
However, different combinations (B1S3, B2S2, B3S1) of SC and BMP improved the overall
359
stability of cheese emulsion against creaming and precipitation. A desirable cheese emulsion
360
stability was obtained by addition of equal amounts of the two ingredients (the B2S2 combination).
361
This might be mainly attributed to the dual effects of SC as restructuring and emulsifying, which is
362
likely to improve in the presence of BMP. Based on the results presented so far, it could be
363
suggested that, to some extent, particle size does not affect stability of cheese emulsion significantly,
364
where fat emulsification and protein stabilization play equally important roles in keeping cheese
365
emulsion stability. Overall, the influence of SC and BMP on rheological properties and stability of
366
hot cheese emulsion may be of practical importance and has potential application as ES replacers.
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367 368
Acknowledgements
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The authors thank the China Scholarship Council (CSC) and Lactosan A/S for providing financial support for this research.
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374 375
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Figure legends
2
Fig. 1. Particle size distribution of seven cheese emulsion samples containing different ingredients
4
(■, control; ●, emulsifying salt; □, sodium caseinate; ○, butter milk powder; ▽, 1% butter milk
5
powder + 3% sodium caseinate; ★, 2% butter milk powder + 2% sodium caseinate; ▼, 3% butter
6
milk powder + 1% sodium caseinate).
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SC
7
Fig. 2. Apparent viscosity variation of cheese emulsions as a function of shear rate (□, control; ●,
9
emulsifying salt; ▲, sodium caseinate; 〇, butter milk powder; ▽, 1% butter milk powder + 3%
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10
sodium caseinate; △, 2% butter milk powder + 2% sodium caseinate; +, 3% butter milk powder +
11
1% sodium caseinate).
12
Fig. 3. Creaming of different samples under centrifugation (□, control; ●, emulsifying salt; ▲,
14
sodium caseinate; ■, butter milk powder; △, 1% butter milk powder + 3% sodium caseinate; ▽,
15
2% butter milk powder + 2% sodium caseinate; ○, 3% butter milk powder +1% sodium caseinate).
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16
Fig. 4. Confocal laser scanning microscopy images of cheese feeds: A, cheese feed containing
18
emulsifying salt; B, cheese feed without any additions; C, cheese feed with 4% w/w (on dry matter
19
basis) added buttermilk powder; D, cheese feed with 4% w/w (on dry matter basis) added sodium
20
caseinate. Representative images are chosen from a series of images available for each sample
21
composition.
AC C
17
1
ACCEPTED MANUSCRIPT Table 1
Control ES BMP SC B1S3 B2S2 B3S1
Abbreviations are: ES, emulsifying salt; BMP, butter milk powder; SC, sodium caseinate; B1S3, 1%
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a
Ingredients (w/w, by dry matter) ES BMP SC Water (g) 117 4% 136 4% 136 4% 136 1% 3% 136 2% 2% 136 3% 1% 136
SC
Sample
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Experimental design and ingredients used.a
butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate;
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B3S1, 1% butter milk powder+3% sodium caseinate.
ACCEPTED MANUSCRIPT Table 2 Physicochemical parameters of the two different types of cheeses. Ash (%)
pH
1.67±0.06 4.48±0.02
3.95±0.02 5.77±0.02
5.46±0.01 4.60±0.00
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pH 4.6 Soluble N (%) 0.66±0.02 0.37±0.01
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Salt (%)
SC
Protein Fat (%) (%) 24.38±0.15 36.8±0.00 15.26±0.18 17.17±0.29
EP
Cheddar Soft white
Moisture (%) 34.28±0.12 60.37±0.13
AC C
Cheese
ACCEPTED MANUSCRIPT Table 3
Control
34.4 ± 4.6a
ES
6.7 ± 1.2c
SC
46.3 ± 7.3a
BMP
24.2 ± 2.2b
B1S3
44.2 ± 9.0a
B2S2
38.4 ± 4.2a
B3S1
26.9 ± 1.7b
SC
D [4, 3] (µm)
Abbreviations are: ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3,1%
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a
Sample
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Average particle sizes of cheese emulsions. a
butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 3% butter milk powder + 1% sodium caseinate. Values are means ± SD (n = 6); means with
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different superscript letters are significantly different (p<0.05).
ACCEPTED MANUSCRIPT Table 4
κ (Pa sn) 0.07 ± 0.02c 2.93 ± 0.42a 0.03 ± 0.02d 0.13 ± 0.04b 0.04 ± 0.02d 0.07 ± 0.06b,c 0.10 ± 0.03b,c
a
n 0.87 ± 0.02b 0.64 ± 0.03d 0.95 ± 0.06a 0.77 ± 0.04c 0.90 ± 0.07a,b 0.88 ± 0.10a,b 0.79 ± 0.03c
R2 0.979 0.967 0.988 0.968 0.978 0.966 0.964
χ2 0.197 26.59 0.014 0.190 0.057 0.155 0.223
SC
Sample Control ES SC BMP B1S3 B2S2 B3S1
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Power Law model parameters.a
Abbreviations are: κ, consistency coefficient; n, flow behaviour index; R2, determination
M AN U
coefficient; χ2, chi-square; ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3, 1% butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 1% butter milk powder + 3% sodium caseinate. Values are means ± SD (n=6); means in the same column followed by different superscript letters are significantly different
AC C
EP
TE D
(p<0.05).
ACCEPTED MANUSCRIPT Table 5 Height of middle phase after centrifugation. a Height (mm)
ES
No observable boundary
Control
25.3 ± 0.6b
SC
18.6 ± 1.1c
BMP
29.7 ± 1.2a
B1S3
19.8 ± 1.8c
B2S2
No observable boundary
B3S1
22.9 ± 1.1b
SC M AN U
a
RI PT
Formulation
Abbreviations are: ES, emulsifying salt; SC, sodium caseinate; BMP, butter milk powder; B1S3,
TE D
1% butter milk powder + 3% sodium caseinate; B2S2, 2% butter milk powder + 2% sodium caseinate; B3S1, 1% butter milk powder + 3% sodium caseinate. Values are means ± SD (n=6);
AC C
EP
means followed by different superscript letters are significantly different (p<0.05).
M AN U
SC
RI PT
Particle volume (%)
ACCEPTED MANUSCRIPT
EP AC C
Fig 1
TE D
Particle diameter (µm)
EP TE D
Fig. 2
AC C M AN U SC
RI PT
Apparent viscosity (Pa s) (%)
ACCEPTED MANUSCRIPT
Shear rate (1/s)
EP
Fig. 3
AC C TE D M AN U SC
RI PT
Height of fat layer (mm)
ACCEPTED MANUSCRIPT
Time (min)
Fig. 4
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT