Physical properties and storage stability of reverse osmosis skim milk concentrates: Effects of skim milk pasteurisation, solid content and thermal treatment

Physical properties and storage stability of reverse osmosis skim milk concentrates: Effects of skim milk pasteurisation, solid content and thermal treatment

Journal of Food Engineering 278 (2020) 109922 Contents lists available at ScienceDirect Journal of Food Engineering journal homepage: http://www.els...

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Journal of Food Engineering 278 (2020) 109922

Contents lists available at ScienceDirect

Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng

Physical properties and storage stability of reverse osmosis skim milk concentrates: Effects of skim milk pasteurisation, solid content and thermal treatment Morten Vormsborg Christiansen *, Troels Bjerregaard Pedersen, Jesper Nagstrup Brønd, Leif H. Skibsted, Lilia Ahrn�e University of Copenhagen, Department of Food Science, 1958, Frederiksberg, Denmark

A R T I C L E I N F O

A B S T R A C T

Keywords: Membrane filtration Rheological behaviour Thermal treatment Particle size Storage stability

Concentrated dairy products are of increasing interest within the dairy industry. Skim milk concentrates can be produced by reverse osmosis membrane filtration, which can be considered a non-thermal process. Therefore, the physical properties of concentrates differ from the properties of concentrates produced by evaporation. In this study, reverse osmosis filtration of raw and pasteurised skim milk was carried out in batch up to 28% total solids content and the effect of thermal treatment (65–110 � C, 15 s) and storage at 5 � C up to ten days on rheological and physical properties of concentrates, were evaluated. Concentrates produced from pasteurised skim milk required longer concentration times and showed larger average casein micelle sizes, but limited structure buildup capability during storage compared to concentrates produced from raw milk which more readily created structural networks between the milk constituents and consequently had higher viscosity. Thermal treatment of concentrates increased their average particle size and viscosity, an effect enhanced by increasing the total solids content. Concentrates produced from non-pasteurised milk showed the strongest shear-thinning behaviour during storage. Thus, the thermal treatment of milk before or after the concentration process, controls the structure formation of skim milk concentrates during storage.

1. Introduction In dairy industry skim milk concentrates (SMC) have various appli­ cations, for example as intermediate products prior to spray drying or as ingredients in the formulation of other products. Recently, on-farm concentration has also been investigated in terms of reduction of transport, cooling and CO2 emission costs (Sørensen et al., 2016). Con­ centration of skim milk can be accomplished by various processes. Evaporation and reverse osmosis (RO) filtration are examples of commonly used concentration technologies aiming increase in total solids (TS) content by removing water. Milk evaporation and drying can be considered the most energy demanding operations of the dairy in­ dustry whereas concentration by membrane filtration demands signifi­ cantly lower amounts of energy (Ramirez et al., 2006). Furthermore, RO can be performed at lower temperatures than evaporation and can therefore be considered a non-thermal concentration process which limits heat-induced whey protein denaturation. Temperature control is on the same time crucial during RO of milk to avoid microbial growth

and biofouling of the membranes. Low processing temperatures are advantageous since the fraction of denatured whey proteins affects the structure of milk concentrates (Sutariya et al., 2017). Nonetheless, concentration by reverse osmosis is not able to reach the same TS level as concentration by evaporation due to restrictions in applicable pressure (Ramirez et al., 2006). In addition to the concentration technology, other parameters such as concentrate TS, storage temperature and time, pH, and heat load before or after the concentration process have showed to influence the final properties of SMC (Anema et al., 2014; Corredig et al., 2019; Gazi and Huppertz, 2015). The protein denaturation rate in concentrated milk systems has been shown to increase with increased thermal treat­ ment, which is explained by the strong influence of temperature on whey protein unfolding (Wolz and Kulozik, 2015). In the same study, thermal denaturation was also found to occur faster with increasing protein concentration probably due to increased whey protein collisions promoting aggregation. It is challenging to produce a dairy concentrate with high stability that maintains low viscosity during storage, as there is a lack of knowledge on how the thermal history of milk affects the

* Corresponding author. Rolighedsvej 26, 1958, Frederiksberg, Denmark. E-mail address: [email protected] (M.V. Christiansen). https://doi.org/10.1016/j.jfoodeng.2020.109922 Received 18 October 2019; Received in revised form 11 January 2020; Accepted 14 January 2020 Available online 16 January 2020 0260-8774/© 2020 Elsevier Ltd. All rights reserved.

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storage in RO milk concentrates, produced from raw and pasteurised skim milk were found. Thus, process conditions that minimise the vis­ cosity of SMC during subsequent storage and transportation would be valuable for industrial handling of concentrates and for development of new products. The objective of the present study is therefore to inves­ tigate whether thermal treatment of skim milk before RO and/or of concentrates after RO can be used to control and maintain stable SMC during storage.

Abbreviations SMC DLS np-SMC p-SMC pdI RO TS TMP

Skim milk concentrates Dynamic light scattering Skim milk concentrates made from non-pasteurised milk Skim milk concentrates made from pasteurised milk Polydispersity Index Reverse osmosis Total solids Transmembrane pressure

2. Materials and methods A schematic drawing summing up the experimental design is shown in Fig. 1. Three independent RO concentration trials were performed between April and August. For each trial, 300 kg pasteurised and 300 kg non-pasteurised bovine skim milk were collected from a local dairy (Slagelse Mejeri, Arla Foods Amba, Denmark) and stored for 20 h at 5 � C before concentration. Non-pasteurised skim milk was collected after the skimming process, which took place at 55 � C in a centrifugal separator, whereas the pasteurised milk was collected after a plate and frame heat exchanger (73 � C, 15 s) placed in connection to the separator. The milk collected directly after the separator is referred to as ‘non-pasteurised’ and the milk collected after the pasteuriser as ‘pasteurised’ skim milk. The composition of the collected milk was analysed on a Milko­ ScanTMFT2 (Foss, Hillerød, Denmark).

properties of milk concentrates, including their rheological properties. Regarding the effects of heating strategies on protein denaturation and structure formation, several studies have investigated both milk and concentrates. For example, the effect of heat treatment by steam injec­ tion on RO-based SMC was investigated by Dumpler and Kulozik (2016), and heating milk by plate and tubular heat exchangers has also been compared with direct steam injection or infusion (Akkerman et al., 2016; Malmgren, 2007). Although the rheological properties of skim milk concentrates produced by evaporation have previously shown to follow power law behaviour (Anema et al., 2014; Debon et al., 2010; Morison et al., 2013; V�elez and Barbosa, 1998), no publications comparing rheological properties and structure formation during

Fig. 1. Schematic representation of the experimental set-up. On the left-hand side where the work was done, in the middle what was done and on the right-hand side the effects studied as reported in the following sections. 2

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2.1. Concentration of skim milk by reverse osmosis

Bars on figures represent standard deviations of triplicate measurements calculated by R software (3.4.2). Variance between groups were ana­ lysed by ANOVA (R 3.4.2).

Milk concentrates with 24 and 28% TS (w/w) were produced batchwise by RO on a GEA pilot scale filtration unit (Skanderborg, Denmark) with a spiral wound type membrane (Dairy-Pro 3838 RO-30, KOCH Membrane Systems, Wilmington). The processing temperature was kept constant at 8 � C during concentration and the transmembrane pressure was increased to 20, 27, 31 and 36 bar when TS contents of 10, 16, 22 and 28% were respectively reached. All milk concentrates were heat treated after concentration at 65, 75, 85, or 110 � C for 15 s on a pilot scale continuous UHT/HTST unit (UHT/HTST Direct & Indirect Pro­ cessing System, Microthermics, Raleigh, USA). Finally, heat treated SMC were stored at 5 � C for up to 10 days and samples were collected along the storage time.

3. Results and discussion RO concentration of skim milk was performed at 8 � C and the change in physical properties during storage at 5 � C were studied for up to 10 days. The effect of pasteurisation of skim milk prior to RO (part 1), the effect of heat treatment of SMC (part 2) and the effect of storage at 5 � C on SMC (part 3) on the efficiency of concentration and/or properties of the concentrates are described. For all replicates the total protein con­ tent and the total solids (TS) content in the collected skim milk was 3.6 � 0.1% and 9.7 � 0.3%.

2.2. Characterisation of skim milk concentrates

3.1. Part 1: milk pasteurisation prior to reverse osmosis filtration

2.2.1. Total solids in skim milk concentrates Total solids in SMC were determined in triplicates according to the reference method (ISO 6731:2010).

3.1.1. Efficiency of the concentration process The concentration process was affected by pasteurisation of the milk prior to RO. Fig. 2 shows that concentration of pasteurised skim milk with an initial TS content of ~10% (w/w) up to a TS content of 28% (w/ w) required approximately 20% longer processing time compared to concentration of non-pasteurised skim milk. The corresponding decrease in flux with increased transmembrane pressure is shown in Fig. 2b. Decrease in flux during filtration of pasteurised milk has previously been described by Hausmann et al. (2013) to be due to concentration and interaction of milk components such as proteins and minerals, which lead to protein and calcium fouling on membrane pores (James et al., 2003).

2.2.2. Rheological properties The rheological properties of SMC were evaluated by a rheometer (Kinexus Pro, Malvern, UK) with a cylindrical rotating bob, D ¼ 35 mm (type C25G WS 1370 SS, Malvern, UK) and a fixed cup (type PCOB25 S1851 SS, Malvern, UK) geometries to determine flow curves over a shear rate (ϒ) range of 10–300 s 1 against applied shear stress (σ). Flow curves were measured at 10 � C. Ten measurements per shear rate decade with a maximum speed of 1.000 rad/s and an acceleration of 10 rad/s2 were recorded. Flow curves were fitted to the power law model

σ ¼ Kγn

3.1.2. Viscosity of non-heated skim milk concentrates Increasing TS increased the apparent viscosity of SMC as displayed in Fig. 3. Removal of water forces the milk constituents closer together and thereby intensify their interactions. The largest viscosity increase was found in SMC produced from non-pasteurised milk (np-SMC) compared to SMC produced from pasteurised milk (p-SMC), implying that changes in milk caused by pasteurisation have an effect on SMC viscosity, especially in SMC with TS above 24% (w/w). Notably the variation between triplicate measurements was larger for np-SMC, in particular for the most concentrated samples (28% TS (w/w)), illustrated by the broader error bars. Pasteurisation of milk has previously been found considerably to increase the heat stability of concentrated milk (Dumpler, 2017; Lin et al., 2018), due to coverage of casein micelles with denatured whey proteins which reduce casein micelle aggregation, leading to lower viscosity. This is in line with Bienvenue et al. (2003), who suggested that the increase in apparent viscosity of SMC prepared from pasteurised milk was due to the combined effects of increased volume fraction and increased particle interactions. It is surprising that the SMC with the higher viscosity (i.e. np-SMC) faster reaches a higher solids content during RO (Fig. 2). This could be due to the fact that np-SMC have stronger shear-thinning behaviour than p-SMC (results presented later). Thus, the high shear rates at the membrane interphase reduce the viscosity, leading to a faster concentration rate. After RO no shear fields were applied, which allowed structure build-up and conse­ quently a higher viscosity is observed in np-SMC. Another effect of pasteurisation is the slight increase in milk pH (Table 1), which is similar to previous reports (ul Haq et al., 2014; Pestana et al., 2015). Further­ more, the pH decreases along the RO concentration, and for p-SMC the pH remains higher than np-SMC when concentrating to 28% TS (w/w). It is important to note, that the observed higher viscosity in np-SMC might also be due to the pH difference between p-SMC and np-SMC, as np-SMC gets closer to the isoelectric point of caseins leading to more interactions and thereby higher viscosity.

(1)

where ϒ is the shear rate, σ the shear stress, n is the flow behaviour index and K the consistency index. For n ¼ 1 the flow behaviour is Newtonian, whereas n < 1 is shear-thinning. Flow curves data were satisfactorily described by the power law model (all r2-values > 0.95, not shown). The Hershel-Bulkley model was also tested, confirming that the SMC did not show yield stress (data not shown). 2.2.3. Particle size distribution Particle size distributions and corresponding z-average values of SMC were measured by dynamic light scattering (DLS) (Zetasizer, Mal­ vern, UK), performed in triplicate. SMC were diluted in RO permeate directly in a 3 mL cuvette (type DT0012, Malvern, Germany). Skim milk (control) samples were diluted ten times whereas SMC were diluted correspondingly to obtain equal TS amounts in all samples (approx. 10%). The temperature was maintained at 23 � C during measurement, the dynamics of the scattering beams were collected at a 12.8� angle and the refractive index of buffer (RO permeate) and milk protein was set to 1.330 and 1.450, respectively. Measurements were carried out imme­ diately after dilution. 2.2.4. pH The pH was measured in the fresh concentrates and during storage using a pH-meter (Delta 320, Mettler Toledo, Columbus, Ohio). The pH meter was calibrated with standard buffer solutions of pH 4.00 and 7.00 (Hamilton, Bonaduz, Switzerland) before use. 2.3. Statistical analysis All experiments were carried out in triplicates i.e. three separate batches were concentrated for both non-pasteurised and pasteurised milk. The influence of the SMC processing parameters pasteurisation, degree of concentration, post-heat, and storage time were evaluated by effect tests on studentised residuals performed in JMP 13 (SAS 9.4 M5).

3.1.3. Particle size of skim milk concentrates Skim milk pasteurisation prior to RO increased markedly the z3

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Fig. 2. Degree of obtained total solids content in skim milk concentrates with processing time (A) and flux through the membrane with transmembrane pressure applied (B). Lines are to guide the eyes.

Fig. 3. Effect on concentrate viscosity with increasing total solid contents on non-heat treated concentrates made from non-pasteurised and pasteurised skim milk. Different superscript letters indicate significant difference (p < 0.05).

average hydrodynamic diameter of particles in p-SMC compared to npSMC (Table 2). A total increase of ~35–40 nm was observed in skim milk from an average size of 130 � 0.2 nm in np-SMC to an average size of 175 � 0.1 nm in p-SMC. In contrast, increasing TS showed slightly decreasing particle sizes in np-SMC (p < 0.05), but this effect was smaller than the effect of thermal treatment (Table 3). The particles measured by DLS were considered to be casein micelles and increasing casein micelle sizes has previously been reported to depend on associ­ ation of denatured whey proteins with the casein micelles (Anema and Li, 2003). Similar effects for the milk particle z-average values and changes with thermal treatments have been reported previously. For example, an average diameter of 169 � 1 nm was reported for pas­ teurised skim milk, followed by a ~40 nm increase when the milk was heated at 120 � C for 10 min (Eshpari et al., 2017). For concentrates,

these results may explain why lower viscosity is observed in p-SMC, since caseins already were associated with denatured whey proteins and less interactions became possible. 3.2. Part 2: heat treatment of skim milk concentrates To inactivate post-contaminating microorganisms a heat treatment of SMC may be needed to extend its shelf-life. In this work, we investi­ gated the physical effects of heat treating SMC at 65, 75, 85, or 110 � C for 15 s, covering heat treatments below, at and above legal re­ quirements. According to the New Zealand Food Safety Authority, dairy materials with greater than 15% TS should be subject to a minimum temperature of 74.9� when a holding time of 15 s is applied (NZFSA, 2009). 4

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concentration and during thermal treatment of concentrates may explain the differences in viscosity between p-SMC and np-SMC. Furthermore, at high TS the collisions between whey proteins and ca­ seins may induce further aggregation and consequently promoting the increase in viscosity. While in p-SMC some casein micelles and dena­ tured whey proteins were previously aggregated due to milk pasteur­ isation prior to RO, less interaction may occur during the post thermal treatment of the concentrates. In np-SMC, however, more casein-whey interactions might be available and prone to take place during thermal treatment. In other words, our results show that more aggregation seems to be promoted at a high TS content in np-SMC compared with p-SMC. As previously discussed, the pH value of SMC to be heat treated might also affect the viscosity, as the pH drops in SMC when the TS content increases (Table 1). This pH-drop is slightly larger in np-SMC compared to p-SMC. Milk pH adjustments before heating has previ­ ously been found to affect whey protein interactions with casein micelles (Anema et al., 2014). They suggested that most of the milk proteins are associated with the casein micelles at pH 6.5, whereas more whey pro­ teins and κ-casein form insoluble aggregates at higher pH. In our case we have a pH decrease from 6.7-6.8 to 6.5 with the increase of TS, which is a small difference but may have promoted interactions.

Table 1 pH of pasteurised and non-pasteurised skim milk and their corresponding con­ centrates heat treated at varying temperatures for 15 s. Based on triplicate measurements. Different superscript letters indicate significant difference (p < 0.05). Skim milk

24% TS (w/w)

28% TS (w/w)

6.58 � 0.02c 6.57 � 0.05c 6.57 � 0.06c 6.56 � 0.04c 6.54 � 0.06c

6.54 � 0.01d 6.54 � 0.03d 6.53 � 0.03d 6.53 � 0.03d 6.51 � 0.01d

milk 6.56 � 0.02c 6.56 � 0.05c 6.55 � 0.06c 6.54 � 0.04c 6.53 � 0.06c

6.49 � 0.01e 6.48 � 0.03e 6.48 � 0.03e 6.48 � 0.03e 6.46 � 0.01e

SMC produced from pasteurised skim milk No heat treatment 65 � C 75 � C 85 � C 110 � C

6.81 � 0.02a 6.84 � 0.02a 6.82 � 0.03a 6.82 � 0.01a 6.80 � 0.03a

SMC produced from non-pasteurised skim No heat treatment 6.73 � 0.02b 65 � C 6.73 � 0.02b 75 � C 6.73 � 0.03b 85 � C 6.72 � 0.01b 110 � C 6.72 � 0.03b

Table 2 Effect on hydrodynamic particle size (z-average) with increasing total solids in milk concentrates based on non-pasteurised (np-SMC) and pasteurised (p-SMC) skim milk. Different superscript letters indicate significant difference (p < 0.05). Total solids [% (w/w)] Skim milk 24 28

3.2.2. Particle size of milk concentrates Thermal treatment of SMC at >75 � C, 15 s, was found to increase the average particle size markedly compared to thermal treatment of unconcentrated skim milk (Fig. 4b). It is assumed that the measured par­ ticle sizes reflect the casein micelles and casein micelles covered by denatured whey proteins. The influence of heat treatment of SMC on casein micelle enlargement depends strongly on whether the milk has been pasteurised or not before RO and on the TS content. The higher protein content in SMC was found to induce significantly larger particle sizes at 28% TS compared with 24% TS both for p-SMC and np-SMC. Similar results has been reported by Wolz et al. (2016). It is assumed that the casein micelle sizes increase as result of heat treatment due to whey protein denaturation with disulphide exposure and covalent bonding to the casein micelle. The increase in particle size in np-SMC due to thermal treatment after concentration is slightly larger compared to p-SMC (Fig. 4b). Np-SMC must contain a larger amount of native whey proteins than p-SMC, and consequently, these results sug­ gest that whey protein denaturation in a concentrated milk matrix leads to more whey protein-casein micelle interactions than when more whey proteins are denatured before milk concentration. More information about the effect of processing parameters on micelle sizes are found when looking at the scattering intensity size distributions (Fig. 5). Intensity size distributions were all unimodal independently of the TS content and thermal treatment of concentrates. This is confirmed by considering the polydispersity index (pdI) based on particle size measurements, which were all < 0.14 (not shown). A pdI <0.2 is normally considered monodisperse (Sinaga et al., 2017). How­ ever, for p-SMC, the size distributions exhibited a broader span. Furthermore, with increasing heating temperatures, the size distribu­ tions became asymmetric to larger particle sizes, an effect enhanced by increasing TS content. The overall monomodality implies no micelle

Z-average [nm] Np-SMC

p-SMC

130.2 � 0.2a 127.5 � 0.5b 127.7 � 0.7b

175.7 � 0.1c 174.5 � 2.3c 172.4 � 1.3c

3.2.1. Viscosity of heat treated skim milk concentrates As expected, SMC viscosity was affected by the degree of concen­ tration and the pasteurisation of milk, but also the intensity of the heat treatment after concentration. The effect of concentration and following heat treatments on SMC viscosity is displayed in Fig. 4a. As previously shown for non-heated concentrates, increasing TS increased the vis­ cosity and this is also observed in heat treated SMC. Thermal treatment of SMC at temperatures >75 � C, 15 s, further increased SMC viscosity, but for skim milk (control), the viscosity was unaffected. These results are in agreement with studies reported by Wolz and Kulozik (2015) and Kieferle et al. (2019), the former suggesting that concentration promotes aggregation by increased collisions between whey proteins. Fig. 4a also shows that the viscosity of 24% TS (w/w) SMC increased similarly for np-SMC and p-SMC. However, when concentrating to 28% TS the vis­ cosity was markedly lower in p-SMC compared to np-SMC. Heat treated casein micelles have previously been reported to introduce chaperon like behaviour on whey protein aggregation in concentrated milk pro­ tein model systems (Liyanaarachchi et al., 2015). As discussed in the previous section, pre-heat treatment changed the casein micelle morphology and probably also their surface properties by interacting with denatured whey proteins. Thus, extension of whey protein dena­ turation and consequently self-aggregation and cross-linking to casein micelles promoted during thermal treatment of the milk prior to

Table 3 Analysis of variance results describing the processing parameter’s effects on the rheological parameters and the particle size in skim milk concentrates. Bold numbers highlight high p-values corresponding to no effect. Source Pasteurisation of milk Heat treatment of SMC Storage time at 5 � C Degree of concentration Pasteurisation*concentration Pasteurisation*SMC heat treatment

Flow behaviour index, n

Consistency index, K

Prob > F

LogWorth

Prob > F

LogWorth

Prob > F

LogWorth

0.09612 <0.001* <0.001* <0.001* <0.001* <0.001*

1.017 8.472 7.880 18.382 24.156 4.110

<0.001* <0.001* <0.001* <0.001* <0.001* 0.6854

4.4972 17.939 4.972 32.914 5.181 0.164

<0.001* <0.001* 0.13343 <0.001* 0.0335* 0.0470*

37.310 30.317 0.875 5.358 1.475 1.328

5

Particle size (z-average)

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Fig. 4. Viscosity measured at shear rate 79.44 Pa (A) and Particle size (B) development as result of heat treatment of pasteurised and non-pasteurised skim milk after reverse osmosis concentration. Bars represent standard deviations of triplicate measurements.

Fig. 5. Intensity size distributions of particles in skim milk concentrates non-pasteurised or pasteurised before RO, and heat treated from none to 110 � C for 15 s after filtration, stored for 1 day at 5 � C. Symbols indicate total solids content (◻ ¼ skim milk, Ο ¼ 24% TS (w/w), and Δ ¼ 28% TS (w/w)).

aggregation in SMC into large aggregates within the storage period and conditions investigated. Conversely to individual micelle enlargement by covalent bonding of denatured whey protein, large casein aggregates are primarily formed by hydrophobic and ionic interactions (V� elez and Barbosa, 1998). They reported bimodal size distribution curves for concentrated milks stored for >8 h at 50 � C with a second peak between 1000 and 5000 nm corresponding to large casein micelle aggregates. In our study, milk was concentrated at low temperatures, and under these conditions the hydrophobic bonds are weakened, which could explain why no large aggregated micelles have been observed.

3.3. Part 3. storage of skim milk concentrates 3.3.1. Rheological properties of skim milk concentrates during storage Changes in rheological properties during storage are important for further handling and processing of the milk concentrates before spray drying, in the formulation of cheese or as ingredient in other products. In this work, the changes in rheological behaviour were studied during storage at 5 � C for up to 10 days. Increasing TS content shifts the flow behaviour from Newtonian to shear-thinning and this effect is enhanced by increasing the heating temperature, as shown in Fig. 6. This is consistent with previous studies on concentrated skim milk at different concentrations (Anema et al., 2014; Morison et al., 2013). The correla­ tion between processing parameters were investigated by a variance analysis, which is summarised in Table 3. Unlike particle size 6

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found to continuously change during storage. In np-SMC the drop in flow behaviour index, n, during storage was faster than in p-SMC (Fig. 6). Pasteurisation before concentration creates less shear-thinning SMC, as also mentioned in the previous section, as seen by flow behaviour index, n. As expected, increasing TS and thermal treatment of SMC increase the consistency coefficient, K. Especially SMC with 28% TS heat treated >85 � C, 15 s showed rapid increase in K during storage. Similar to the flow behaviour index more dramatic changes in K occurs in np-SMC compared to p-SMC. It is concluded from the results discussed above that the cumulative effect of heat load and storage time is substantially different between pSMC and np-SMC. More structure is developing in np-SMC, whereas prepasteurisation of milk seems to have a stabilising effect. It could be of interest to investigate whether this difference is maintained during further storage than 10 days or if the difference levels off over time. 3.3.2. Particle size of skim milk concentrates during storage Contrary to viscosity, the storage time of up to 10 days at 5 � C was found not to have any significant effect on the Z-average in SMC as shown in Table 3. The monomodal shape of the particle size distribu­ tions remained during storage at all heat loads applied to SMC. Fig. 5 displays representative size distribution curves for SMC after 1 day storage at 5 � C. The np-SMC in general exhibit sharper size distributions and lower amounts of large particles compared to p-SMC. Skim milk concentrate viscosity has previously been reported to be governed pri­ marily by particle interactions rather than distribution of denatured proteins in milk serum (Sutariya et al., 2017). The particles created by an overall more severe heat load in p-SMC heated >85� , 15 s, are probably less interactive than the corresponding particles in np-SMC. However, this data should be carefully interpreted because the dilu­ tion of SMC prior to DLS measurements may disrupt weak aggregates that may have been formed. Thus, aggregates formed in np-SMC could be formed by weaker bonds compared to p-SMC, but this needs to be further investigated. Opposite to other studies, the undesired age-gelation and sedimen­ tation previously reported for milk concentrates prepared by other means than RO and stored for longer times and at higher temperatures (Bienvenue et al., 2003; Cao et al., 2016; V�elez and Barbosa, 1998) have not been observed in the present study. It is generally recognised that casein micelle sizes increase during storage but at a relatively low rate (at low temperatures) (Gaucher et al., 2008; Malmgren et al., 2017). The asymmetry observed in Fig. 5 towards larger particle sizes for >5 days of storage confirms that casein micelles interact, but only slowly during storage at low temperatures.

Fig. 6. Flow behaviour index, n, and consistency coefficient, K, for nonpasteurised (A) and pasteurised (B) milk based SMC as function of length of storage at 5 � C. Symbols indicate heat treatment of SMC: ◻, Ο, Δ, ☆, and þ indicate no heat, 65 � C, 75 � C, 85 � C, and 110 � C respectively, for 15 s.

development, the effect of pasteurisation before concentration could not explain the observed change in flow behaviour (p ¼ 0.096). However, the combined effect of pasteurisation and increased TS during concen­ tration showed the largest total effect on the flow behaviour. In other words, pasteurisation prior to concentration seems to slow down the structure formation responsible for shear-thinning behaviour during storage. Dumpler (2017) reported that the heat stability, evaluated by gelation and sediment formation, progressively decreased with increasing TS contents in SMC produced by RO, but appropriate pre­ heating of milk before concentration increased the heat stability of milk concentrates. High pressures applied to milk has a disruptive effect on casein micelles (Needs et al., 2000), including the pressure applied during RO filtration (Corredig et al., 2019). Interactions between de­ natured whey proteins and caseins in either intact or pressure-disrupted states (as in pasteurised milk or np-SMC, respectively) may be different. Even though the pressure-induced disruption is partially reversible (Tromp et al., 2014), the different amounts of denatured whey proteins in pasteurised and non-pasteurised skim milk and following different binding patterns to caseins could explain why different rheological be­ haviours are observed between p-SMC and np-SMC during storage. Rheological behaviour of SMC induced by thermal treatment was

4. Conclusion Raw and pasteurised skim milk were subjected to concentration by RO and the physical properties of the concentrates were evaluated during storage for up to 10 days at 5 � C. Pasteurisation prior to con­ centration increased the concentration processing time and the average casein micelle size, but decreased the capability of structure build-up during storage. Thus, RO concentrates produced from non-pasteurised skim milk tend to more easily create structural networks between the milk constituents during storage. In pasteurised milk denatured whey proteins are already associated with the casein micelles, leading to less interactions and lower viscosity compared to SMC produced from nonpasteurised skim milk. Thermal treatment after RO concentration caused increasing particle size and viscosity in concentrates and the effect of such thermal treatment was enhanced by increasing total solids content. During storage at 5 � C for up to 10 days, structure was slowly built-up in the concentrates resulting in decreasing flow behaviour index, n, and increasing consistency coefficient K. Skim milk concen­ trates showed shear-thinning behaviour during storage, whereas the size of casein micelle particles only slightly changed during storage. These results are important to improve the handling and processing of the skim 7

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milk concentrates by reverse osmosis, in order to reduce energy con­ sumption and produce more functional dairy products.

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Author contribution section Morten Vormsborg Christiansen: Methodology, Formal analysis, Investigation, Visualization, Writing – Original Draft Troels Bjerre­ gaard Pedersen Methodology, Investigation Jesper Nagstrup Brønd Methodology, Investigation Leif H. Skibsted Supervision, Writing – � Conceptualization, Supervision, Review and Editing Lilia Ahrne Writing – Review and Editing. Declaration of competing interest None. Acknowledgements The authors wish to thank Sylvain Barjon and Nils Mørk for their support in setting up the trials at the pilot plant. We also thank Fran­ ciscus Winfred J van der Berg for statistical discussions. Furthermore, Slagelse Mejeri who provided the skim milk is gratefully acknowledged. The present study is part of the Platform for Novel Gentle Processing, finansially supported by the Dairy Rationalization Fund (DDRF), Uni­ versity of Copenhagen and Arla Foods Amba. References Akkerman, M., Rauh, V.M., Christensen, M., Johansen, L.B., Hammershøj, M., Larsen, L. B., 2016. Effect of heating strategies on whey protein denaturation—revisited by liquid chromatography quadrupole time-of-flight mass spectrometry. J. Dairy Sci. 99 (1), 152–166. https://doi.org/10.3168/jds.2015-9924. Anema, S.G., Li, Y., 2003. Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size. J. Dairy Res. 70 (1), 73–83. https://doi.org/10.1021/jf062734m. Anema, S.G., Lowe, E.K., Lee, S.K., Klostermeyer, H., 2014. Effect of the pH of skim milk at heating on milk concentrate viscosity. Int. Dairy J. 39 (2), 336–343. https://doi. org/10.1016/j.idairyj.2014.08.010. Bienvenue, A., Jim� enez-Flores, R., Singh, H., 2003. Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage. J. Dairy Sci. 86 (12), 3813–3821. https://doi.org/10.3168/jds. S0022-0302(03)73988-5. Cao, J., Wang, G., Wu, S., Zhang, W., Liu, C., Li, H., Li, Y., Zhang, L., 2016. Comparison of nanofiltration and evaporation technologies on the storage stability of milk protein concentrates. Dairy Sci. Technol. 96 (1), 107–121. https://doi.org/10.1007/s13594015-0244-3. Corredig, M., Nair, P.K., Li, Y., Eshpari, H., Zhao, Z., 2019. Invited review: understanding the behavior of caseins in milk concentrates. J. Dairy Sci. 102 (6), 4772–4782. https://doi.org/10.3168/jds.2018-15943. Debon, J., Prud^encio, E.S., Petrus, J.C.C., 2010. Rheological and physico-chemical characterization of prebiotic microfiltered fermented milk. J. Food Eng. 99 (2), 128–135. https://doi.org/10.1016/j.jfoodeng.2010.02.008. Dumpler, J., 2017. Heat stability of concentrated milk sytems. Retrieved from. https://li nk.springer.com/content/pdf/10.1007%2F978-3-658-19696-7.pdf. Dumpler, J., Kulozik, U., 2016. Heat-induced coagulation of concentrated skim milk heated by direct steam injection. Int. Dairy J. 59, 62–71. https://doi.org/10.1016/j. idairyj.2016.03.009. Eshpari, H., Jimenez-Flores, R., Tong, P.S., Corredig, M., 2017. Thermal stability of reconstituted milk protein concentrates: effect of partial calcium depletion during membrane filtration. Food Res. Int. 102 (March), 409–418. https://doi.org/ 10.1016/j.foodres.2017.07.058.

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