Innovative Food Science and Emerging Technologies 34 (2016) 68–76

Contents lists available at ScienceDirect

Innovative Food Science and Emerging Technologies

j ourna l homepage: www.e lsev ie r .com/ locate / i fset

Impact of cryoconcentration on casein micelle size distribution, micellesinter-distance, and flow behavior of skim milk during refrigerated storage

Alseny Balde a,b, Mohammed Aider a,c,⁎a Institute of Nutrition and Functional Foods (INAF), Université Laval, Quebec G1V0A6, Canadab Department of Food Sciences, Université Laval, Quebec G1V0A6, Canadac Department of Soil Sciences and Agri-Food Engineering, Université Laval, Quebec G1V0A6, Canada

⁎ Corresponding author at: Department of Food ScieG1V0A6, Canada. Tel.: +1 418 656 2131 #4051; fax: +1

E-mail address: mohammed.aider@fsaa.ulaval.ca (M. A

http://dx.doi.org/10.1016/j.ifset.2015.12.0321466-8564/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 October 2015Received in revised form 30 December 2015Accepted 31 December 2015Available online 27 January 2016

Cryoconcentration combinedwith a cascade effectwas used to concentrate skimmilk up to 25.12% total drymat-ter. Size, shape, and inter-micellar distance of caseinmicelles were characterized by ZetasizerNano-ZS, transmis-sion electron microscopy, and ImageJ analyses. Flow properties of the cryoconcentrated skim milk wereevaluated during 5weeks of storage under refrigerated condition at 4 °C. Milk color was also evaluated accordingto the L*, a*, and b* system. The cryoconcentrated skim milk obtained after three cryoconcentration cycles wascharacterized by a monomodal distribution of its micelles with a tendency to smaller casein micelles. Approxi-mately 60% of the total micellar volume was occupied by the casein micelles with a size of 100–200 nm, lessthan 18% of the volume with a size of 50–100 nm and only less than 1% was occupied by micelles with a sizeN350 nm. This result shows that cryoconcentration changed the distribution of the mean size of the casein mi-celles to smaller units. No significant difference was observed on the inter-micellar distance. Cryoconcentrationsignificantly improved the color of skim milk by increasing the L* value up to 67 which was similar to that ofwholemilk. Transition from aNewtonian to a non-Newtonian behaviorwas observed from the fourthweek stor-age with a slight increase of casein micelle size.Industrial relevance: A concentration procedure of skimmilk based on a complete block cryoconcentration tech-nique was proposed. Application of this sub-zero technology permitted the concentration of skimmilk total drymatter up to 25%. The caseinmicelle sizewas positively affected bymoving themajor part of themicelles towardthe smaller size, whereas the inter-micellar distance was not affected. This new knowledge can be exploited inmilk-based products to enhance the product stability. The cryoconcentrated skim milk color was positivelyaffected since its L* value, which represents the milk whiteness, was significantly improved. The flow behaviorof the cryoconcentrated milk was of Newtonian type up to 4 weeks of storage at 4 °C. The generated knowledgein this study can be easily used by the milk processing industry in order to make stable milk product with highdrymatter content without addingmilk powder, which negatively affects the product sensory properties (flouryconsistency).

© 2016 Elsevier Ltd. All rights reserved.

Keywords:CryoconcentrationSkim milkCasein micellesSizeRheologyColor

1. Introduction

The use of concentration processes in dairy industry can significantlycontribute to enhance the overall efficiency of milk processing sincehuge quantities of milk can be reduced by concentrating the total drymatter of some specific components such as proteins, yielding advan-tages in terms of processing, packaging, transportation, and handlingKeshani, Luqman Chuah, Nourouzi, Russly, & Jamilah, 2010. The selec-tion of a convenient concentration process depends on the requiredlevel of concentration, the impact of the process on products quality,

nces, Université Laval, Quebec418 656 3723.ider).

available energy resources, and the relative cost of the process. Some-times, a combination of different concentration processes is also used(Morison & Hartel, 2006). Currently, there are several concentrationmethods available for enhancing milk concentration such as vacuumevaporation, reverse osmosis, ultrafiltration, and cryoconcentration inits different variants (freeze concentration) Miyawaki, Liu, Shirai,Sakashita, & Kagitani, 2005. Cryoconcentration of skimmilk is a processof concentrating the solid matter contained in a the aqueous phaseby removing part of water in a form of ice Aider, de Halleux, &Melnikova, 2009. The ice formation can be achieved by different wayssuch as suspension crystallization, progressive freeze concentration,and complete block cryoconcentration Gunathilake, Dozen, Shimmura,& Miyawaki, 2014; Iritani, Katagiri, Okada, Cao, & Kawasaki, 2013.Among these techniques, the complete block cryoconcentration is the

69A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

simplest one formilk concentration and it is based on a controlled freez-ing followed by a controlled passive or assisted thawing.

This technology, being operated at sub-zero temperature conditions,is attractive for heat sensitive liquid foods since it allows retaining thenutritional quality and aromatic compounds within the product. Thisparticularity is attributed to the low operating temperatures, whichare suitable to avoid the degradation of the sensitive liquid food compo-nents such as heat labile proteins, vitamins, and volatiles (Flesland,1995; Ghizzoni, Del Popolo, & Porretta, 1995, and the absence of a liq-uid–vapor interface. Actually, it is regarded as highly promising separa-tion process ofwater from liquid foodswithout affecting the quality andproperties of other components (Fellows, 2000). Considering theseadvantages, cryoconcentration technology has been investigated byseveral researchers for its application to a variety of liquid foods, suchas milk, milk whey, fruit juices, maple sap, and saline solutions DiCesare, Cortesi, & Martini, 1993. Cryoconcentrated milk is a dairy prod-uct that may be utilized as an intermediate material for sterilized,sweetened condensed milk, and Greek-type yoghurt production or asa final product for the consumer or use in different food formulations.

However, even if cryoconcentration technology seems to offer sever-al advantages in comparison with other concentration techniques suchas heat evaporation and membrane concentration (Thijssen, 1970;Thijssen & Van Der Malen, 1981), a high dehydration of casein micellesby water removal processes is of particular importance (De Kruif, 1999;Morris, Foster, & Harding, 2000;Walstra, 1979), since it can increase thevolume fraction of dispersed particles and the inter-micelles interac-tions Bienvenue, Jimenez-Flores, & Singh, 2003. The latter (inter-mi-celles interactions) is very important since the casein micelles havethe greatest impact on the milk macroscopic and functional propertiesLiu, Dunstan, & Martin, 2012, and they are the main contributors tothe viscosity of milk (Walstra & Jenness, 1984) and significantly influ-ence the cheese yield. Thus, any factor that alters the aggregation stateof casein micelle, such as pH, concentration, and salt balance, undoubt-edly affect the viscosity of milk (Bienvenue et al., 2003). In skim milk,the continuous phase viscosity is largely determined by lactose concen-tration, whereas the volume fraction of suspended material is deter-mined by proteins such as casein micelles, dissociated caseins, nativewhey proteins, and denatured whey proteins (Anema, 2008; Jeurnink& De Kruif, 1993). It has been reported that when cryoconcentration isused, the increase of the total drymatter in the concentrated phase is ac-companied by an increase of the amount of lactose entrapped in the icecrystals (Aider & Ounis, 2012). However, little is currently known howthe physico-chemical properties of the casein micelles change in re-sponse to cryoconcentration.

A better understanding of the physico-chemical properties ofcryoconcentrated milk and the changes occurring with progressivelyincreasing cryoconcentration level is needed. This is necessary tofurther understand the dynamics of structure changes duringcryoconcentration and ultimately better determine the main princi-ples ruling the processing of cryoconcentrated milk under differentstorage conditions. Many studies have been conducted on the viscos-ity of concentrated milk prepared by heat evaporation (Vélez-Ruiz &Barbosa-Cánovas, 1998), ultrafiltration Karlsson, Ipsen, Schrader, &Ardö, 2005, or powder reconstitution Alexander, Rojas-Ochoa, Leser,& Schurtenberger, 2002; Anema, 2008; Dahbi, Alexander, Trappe,Dhont, & Schurtenberger, 2010, and some flow properties of freeze-concentrated skimmilk were reported (Chang & Hartel, 1997). By con-trast, limited information is available on the effects of freezing proce-dures such as the cryoconcentration on the micelle size, the inter-micellar distance (spacing between casein micelles), and the productflow properties during storage.

Hence, the purpose of this study is (1) to evaluate howcryoconcentration combined with a cascade affect influences thesize of casein micelles, the inter-micelle distance, as well as thecryoconcentrated skim milk color, and (2) to establish the impactof the cryoconcentration procedure on the rheological properties of

cryoconcentrated skimmilk during refrigerated storage. The evaluationof the influence of temperature and concentration on the apparent vis-cosity was also performed during 5 weeks of storage at 4 °C.

2. Materials and methods

2.1. Skim milk and cryoconcentration procedure

Pasteurized skimmilk was purchased from Natrel (Agropur Cooper-ative, Quebec, Canada) and was used as the start material. Skim milkproximate composition was the following: total dry matter, 9.24 ±0.15%; lactose, 4.91 ± 0.21%; total protein, 3.54 ± 0.17%; and ash con-tent, 0.79 ± 0.11%. The initial pH value was 6.5 ± 0.15.

The cryoconcentration procedure was carried out by applying thecascade principle (effect) as reported by Aider and Ounis (2012). Todo this, glass bottles of 9.3 cm inner diameter and 15 cm height wereused. The bottles were filled with 1000 ml skim milk and were frozenin a freezer at −20 ± 2 °C. After 24 h of freezing, the thawing stepwas carried out under simple gravitational separation of the concentratefrom the ice block at ambient temperature (23 ± 1 °C). The collectedfraction was maintained at near-zero temperature by immersing thecollection bottle in ice water. This procedure avoided any risk of bac-terial growth during the thawing period. At this step, 500 mL of theinitial frozen volume was thawed and collected (50% of the initialvolume). This fraction constituted the concentrated phase of the1st cryoconcentration cycle. The same procedure was repeated withthe collected concentrated solution, which was used as a feed solu-tion for the 2nd cryoconcentration cycle. At the end of the secondcryoconcentration cycle, 50% of the thawed solution was collected.This procedure was repeated at the 3rd cryoconcentration cycles.Each concentrate at a given cycle was used as feed solution for thenext cycle. To avoid any bacterial growth during the storage of the col-lected samples, sodium azide (0.01% w/w) was added to the skim milkas a preservative.

2.2. Analyses

2.2.1. Total dry matterTotal dry matter for all samples was determined by measuring

weight loss upon drying in an oven at 100 °C under vacuum until con-stant weight (24 h) and expressed as dry matter content/total weightin %. The accuracy of the measurements was verified by a freeze dryingcontrol of some samples. No noticeable differencewas find between themeasurements.

2.2.2. Total proteinsTotal protein content of each sample was determined by Dumas

combustion method by using an FP-528 Leco apparatus (Leco Corpo-ration, St. Joseph, MI, USA). The instrument was calibrated withethylenediaminetetraacetic acid (EDTA) as a nitrogen standard. Thepercentage of total protein content was calculated from nitrogencontent by multiplying by a factor 6.38 (IDF, 2002).

2.2.3. Ash and mineral fraction analysisAsh content of different skim milk samples and ice fractions was

measured by the incineration method in a muffle furnace at 550 °C for20 h. The specific mineral analysis (Ca, P, Mg, Na, and K) was carriedout by the inductively coupled plasma method (ICP, Optima 4300 DV,Perkin-Elmer, Norwalk, CT, USA) with the following wavelengths:317.933, 396.847, and 393.366 nm for Ca; 285.213, 280.271, and279.553 nm for Mg; 766.490 nm for K; and 589.592 and 588.995 nmfor Na (Carnovale, Britten, Couillard, & Bazinet, 2015).

2.2.4. Concentration factor and process efficiencyThe concentration factor for each component (proteins, ash, specific

minerals) at each cryoconcentration cycle was calculated as a ratio of its

70 A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

content in the cryoconcentrated fraction at a given cycle (Cn) to its con-tent in the initial feed skimmilk solution (C0) by the following equation(Eq.(1)).

C f ¼Cn

C0� 100% ð1Þ

where Cf is the concentration factor for specific component (%), Cn is thecorresponding concentration at a given cryoconcentration cycle (n=1,2, and 3), and C0 is the specific component concentration in the initialskim milk solution.

Cryoconcentration process efficiency was calculated by the follow-ing equation (Eq.(2)):

PE ¼ DM nð ÞDM n−1ð Þ

� 100% ð2Þ

where DM(n) is the total dry matter content at a given cryoconcentrationcycle, and

DM(n-1) is the total dry matter corresponding to the precedingcryoconcentration cycle.

Eq. (2) was adapted to calculate the process efficiency for each com-ponent by replacing the DM(n) and DM(n-1) by the concentration of eachtargeted component (proteins, ash, and specific minerals).

The recovery efficiency of total dry matter, proteins, and specificminerals was calculated as follows (Eq. (3))

Reff ¼Ci � Ci�1

Ci�1∙100% ð3Þ

where Ci is the concentration of the targeted component at the ithcryoconcentration cycle (i = 1, 2, 3), and C(i − 1) is its concentration inthe previous cycle.

2.2.5. Color measurementThe color parameter measurements of the initial and

cryoconcentrated skim milk were carried out according to themethod reported by Hwang et al. (2007), after calibrating its originalvalues with a standard plate (Y = 92.6, x = 0.3162, y = 0.3323). L*,a*, and b* values were regarded as indicator of lightness, redness, andyellowness, respectively. A volume of about 12.5 ml of milk was placedin a glass cuvette, inserted into a black chamber (provided by KonicaMinolta), and connected to the chromameter. The color of each milksample was determined at room temperature using a chromameter(Konica Minolta CR-300, Morinuchi, Tokyo, Japan) and recorded in theCIE-Lab tristimulus system. Each measurement was taken in triplicatefor each sample and average values were used.

2.3. Transmission electronic microscopy

After a cryoconcentration procedure was completed, a small drop(50 μL) of each sample was immediately fixed by adding 2.5% glutaral-dehyde in cacodylate buffer (0.1M, pH 7.3) for 24 h at 4 °C. The samples

Table 1Proximate composition of initial and cryoconcentrated skim milk and process efficiency as a fu

Component Initial skim milk Concentration cycle

1st 2nd

Total solids (w/w) 9.24 ± 0.01 14.73 ± 0.03 21.36 ± 0.04Total Ca, mg/100 g 107.3 ± 1.6 175.9 ± 2.8 249.1 ± 3.9Total P, mg/100 g 84.7 ± 2.2 139.4 ± 3.8 197.2 ± 4.7Total K, mg/100 g 97.2 ± 1.7 167.1 ± 3.0 250.1 ± 6.0Total Na, mg/100 g 54.1 ± 1 87.4 ± 1.7 119.6 ± 2.9Total Mg, mg 100 g 9.1 ± 0.2 15.2 ± 0.4 22.4 ± 0.7Protein, % (w/w) 3.54 ± 0.17 5.66 ± 0.07 7.73 ± 0.24Ash, % (w/w) 0.72 ± 0.01 1.4 ± 0.01 1.52 ± 0.02

were then dehydrated with serial concentrations of ethanol (30%, 50%,70%, 95%, and 100%). The samples were embedded in Epon, sectionedwith a diamond knife in an ultra-microtome (60–80 nm), stained withuranyl acetate (3%) and lead citrate (0.1%), and observed by using atransmission electron microscopy (JEOL JEM-1230 JEOL Ltd., Tokyo,Japan) at 80 kVwith amagnification factor of 12,000withGatanMicros-copy Suite (Digital Micrograph software, Version 2.11.1404.0). Untreat-ed milk was observed with the same manner and used as a controlsample.

2.4. Image analysis

For each sample, at least 20 transmission electronmicrographsweretaken. The distance between the caseinmicelles wasmeasured by usingImageJ software (ImageJ, Version 1.48v). The mean values of the dis-tance measured at five different sides were taken as representativevalues of each sample. All calculated values were statistically analyzedand validated.

2.5. Particle size determination

The average particle size of casein micelles was measured by usingthe Malvern ZetasizerNano-ZS instrument (Malvern Instruments Ltd.,Worcestershire, UK). The instrument is equipped by a laser with awavelength of 633 nm. The intensity of scattered light is detected atthe 173° angle. Cryoconcentratedmilk from different cycles was diluted200 fold and filtered (0.45 μm nylon filters). Milk permeate was ana-lyzed. A volume of 1500 μL of each sample was inserted into a single-use disposable sizing cuvette DTS0012 following the method reportedin the work of Mootse et al. (2014). The light-scattering measurementswere conducted at 25 °C and initiated 4 min after dilution. All measure-ments were performed in triplicate.

2.6. Rheological measurements

The rheological measurements were conducted with a PhysicaRheometer (Physica-Rheolab ARES-G2, TA Instruments, Wood Dale,IL, USA) by using concentric cylinder (DIN) geometry. Experimentalstudies were conducted on skim milk. Skim milk with 3.54% proteinand 90.75% moisture was used to make concentrates of 14.76%,21.36%, and 25.12% total dry matter for rheometric measurementsat 5 °C, 15 °C, and 25 °C during 5 weeks of storage at 4 °C. The threeselected temperatures represent refrigeration, intermediate, and at-mosphere conditions, respectively, as possible temperatures inwhich the concentrated milk may be stored and handled by indus-tries or by the consumers in home conditions. An aliquot volume of12.5 mL sample of cryoconcentrated milk was loaded in the cup ofRheometer. The sample was pre-sheared at 500 s−1 for 60 s, andshear rate was monitored over a range of 10–1000 s−1 during12 min.

nction of the cryoconcentration cycle.

Process efficiency, %

3rd 1st 2nd 3rd

25.12 ± 0.12 159.42 ± 31 231.17 ± 35 271.86 ± 42325.9 ± 5.0 163.93 ± 27 232.15 ± 31 303.73 ± 15249.9 ± 6.9 164.58 ± 18 232.84 ± 19 295.04 ± 23332.3 ± 6.3 171.91 ± 15 257.30 ± 13 341.87 ± 11137.4 ± 2.6 161.55 ± 17 221.07 ± 13 253.97 ± 1928.6 ± 0.4 167.03 ± 20 246.15 ± 27 314.29 ± 129.02 ± 0.13 159.89 ± 7 218.36 ± 12 254.80 ± 181.94 ± 0.12 194.44 ± 9 211.11 ± 13 269.44 ± 11

Table 2Color parameters of the cryoconcentrated skim milk.

Colorparameter

Initial skim milk Concentration cycle

1st 2nd 3rd

L* 62.68 ± 0.25a 66.32 ± 0.75b 66.96 ± 0.16b 66.97 ± 0.36b

a* −4.98 ± 0.04c −6.1 ± 0.12c −6.63 ± 0.04c −7.37 ± 0.07c

b* −3.525 ± 0.02d 0.44 ± 0.13e 1.58 ± 0.08e 2.67 ± 0.08e

Data are presented as mean ± standard deviation.

64

7267

6165

42

5047

3641

30 3228

15

26

0

20

40

60

80

100

120

Ca K Mg Na P

Eff

icie

ncy

of t

he m

ain

min

eral

s re

cove

ry, %

Type of the targeted minerals

C0

C1

C2

C3

Fig. 2. Effect of the cryoconcentration on the recovery of differentmilkminerals calculatedby the equation (Eq. (3)).

71A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

Both simple power law and Herschel–Bulkley models were used todescribe the shear rate–shear stress data for high-concentration skimmilk:

Power‐law : σ ¼ Kγ: n ð4Þ

Herschel–Bulkley models : σ ¼ σ0 þ Kγ: n ð5Þ

where σ is the yield stress, K is the consistency index (N sn/m2), and n isthe flow behavior index.

2.7. Statistical analysis

All experiments had three replications for each treatment and mea-surement. Data were presented asmeans and standard deviations (SD).Analysis of variance (ANOVA) was performed, and the mean compari-sons were carried out by Tukey's test at 95% confidence level. Statisticalanalysis was performed using the SAS 9.3 software (SAS Institute Inc.,Cary, NC, USA).

3. Results and discussion

3.1. Dry matter, total protein content, and concentration factor

The chemical composition of the initial skim milk andcryoconcentrated milk fractions is summarized in Table 1. The ini-tial total dry matter content of the skim milk was 9.24% ± 0.01%.By increasing the cryoconcentration cycle, the total dry matter con-tent increased approximately linearly and reached 14.73% ± 0.03%,21.36% ± 0.04%, and 25.12% ± 0.12% at the end of the first, second,and third cryoconcentration cycle, respectively. The evolution of thetotal dry matter can be described by a linear regression equation asfollows:

TDM ¼ 5:4243� CrSþ 4:0539 ð6Þ

with R2 = 0.9895 and where TDM is the total dry matter (%) and CrS isthe cryoconcentration cycle.

Fig. 1. Casein micelles of the initial skim (9.25% DM) (a) and cryoconcentrated skim milkcryoconcentrated skim milk was fixed in glutaraldehyde at 20 °C.

These results are in agreement with those reported by Aider et al.(2009). The highest process efficiencies were recorded at the end ofthe first and second cryoconcentration cycles. This result indicatedthat the recovery of the total dry matter was the highest at these cycles.However, the results obtained in the present study showed smaller drymatter recovery compared to the results reported in the study of Aiderand Ounis (2012), who reached higher concentration levels (Aider &Ounis, 2012). This quantitative differences could be attributed to somehandling procedures and the geometry of the used devices. The initialtotal protein content of the skim was 3.54% ± 0.17%. By increasing thecryoconcentration cycle, the total protein content increased in a quasi-linear mode and reached average values of 5.66% ± 0.07%, 7.73% ±0.24%, and 9.02% ± 0.12% and the end of the 1st, 2nd, and 3rdcryoconcentration cycle, respectively. These values represent an in-crease of protein content of 68.4, 145, and 233% at the first, second,and third cryoconcentration cycle, respectively, compared with the ini-tial total protein content.

3.2. Color parameter measurements

Comparisons of the color parameters between cryoconcentratedmilk samples and the control are shown in Table 2. The L* value is ameasure of milk whiteness, a* is the measure of milk color indicatinga tendency to the greenish (if negative) or redness if (if positive),while the b* value is the measure of milk blueness (if negative) oryellowness (if positive). As it can be observed on the obtained data,the milk whiteness increased significantly between the control (initial

(25.12% DM) (b) observed in transmission electron microscopy (TEM). The undiluted

Fig. 3. Particle size distributions of the initial skim milk (C0) and cryoconcentratedskim milk at different cryoconcentration cycles: C1, C2, and C3: 1st, 2nd, and 3rdcryoconcentration cycle, respectively.

72 A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

skim milk) and the cryoconcentrated milk after the 1st cycle 1(P b 0.001), but no significant difference was detected between thewhiteness at the three cryoconcentrated cycles. Negative values of thea* parameter indicate thatmilks are greener rather than red. All samplesare greener, and no significant difference was detected between thecryoconcentrated milks and the control. Positive b* values indicateyellowness and negative values indicate blueness. The obtained resultsin the present study showed that milk yellowness increased as thecryoconcentration cycle was increased, and all cryoconcentratedsamples were significantly different from the control (P b 0.001).These observations are in agreement with the study of Quinoneset al. (1997), who reported that an increase of protein content in-creases whiteness and yellowness but decreases the greenness ofmilk. According to Philips et al. (1995), the L* value of milk hasbeen demonstrated to have the most positive impact on consumerappeal. Previous reports have shown that consumers have thehighest appeal for milks with visual properties close to the wholemilk (Owens, Brewer, & Rankin, 2001). Accordingly, we suggest thatthe process of skim milk cryoconcentration enables making white-yellowish milk, which can preserve the similar appearance of non-thermal treated fresh whole milk.

3.3. Casein micelle shape

Observation of the caseinmicelles from the initial skimmilk showeda roughly spherical shapewith various sizes (Fig. 1a). The appearance ofcasein micelles of the cryoconcentrated skim milk until 25.12% (w/w)total dry matter also exhibited nearly spherical shapes with a widerange of sizes (Fig. 1b). This appearance is in agreement with previousreports of Srilaorkul et al. (1991) on skim milk concentrated 5-foldby ultrafiltration. The structures of caseinmicelles appear as dark cir-cular close-packed. In the case of our study where skim milk wascryoconcentration until 25.12% dry matter, there were no signs ofpronounced aggregation or ofmerged caseinmicelles. Cryoconcentrationdid not result in a significant reduction of the distance betweencasein micelles, which varied from approximately 160 nm in the

Table 3Size distribution (%) of casein micelles in skim milk and cryoconcentrated milk.

Cryoconcentration cycle Particle size of casein micelles (nm)

50–100 100–150

0 (initial) 10.477 35.671st 17.834 36.992nd 16.11 38.1473rd 18.388 39.23

unconcentrated skim milk to 139 nm in the concentrated up to25.12% (w/w) total dry matter. In the literature, the study of Karlssonet al. (2005) reported different results, but the authors concentratedskim milk by ultrafiltration. This difference suggests that the actionof cryoconcentration on skim milk casein micelles may be quite dif-ferent then the effect of pressure driven concentration process suchas ultrafiltration.

3.4. Cryoconcentration effect on soluble calcium and casein micelle sizedistribution

Cryoconcentration process of skim milk resulted in a slightly lowerpH (6.5) compared to the non-concentrated skim milk pH (6.7). Thischange of pH was accompanied by minerals concentration in theconcentrated milk. However, cryoconcentration efficiency decreaseddepending on the number of cycles used by 2/3 of Mg, K, P, and lessthan 2/3 of Ca and Na in the concentrated part at the first cycle andless than 50% and 30% at second and third cycle, respectively (Fig. 2).

Theparticle size distributions in skimmilk concentrates as a functionof the cryoconcentration cycle is shown in Fig. 3. Cryoconcentratedmilk casein micelle size distribution was monomodal, with all themicelle size ranging from 50 to 350 nm. It has been observed that in-creasing the concentration cycle tends to reduce themicelle size. Thecryoconcentration process caused a significant decrease (P b 0.001)of the average casein micelle size, which ranged from 166.5 nm atthe 1st cryoconcentration cycle to 152 nm at the 2nd cycle and147.9 nm at the 3rd cycle. The reason for this decrease is likely anoverall contraction,which affects the balance ofminerals, especially cal-cium and phosphor, as shown by the linearly increased (R2 = 0.991)mineral content in the soluble fraction. The size distribution of caseinmicelles obtained from the control (9.25% DM), cycle 1 (14.73% DM),cycle 2 (21.36% DM), and cycle 3 (25.12% DM) are presented inTable 3. In this study, casein micelles were classified into 7 classes ac-cording to 50 nm increments. Micelles larger than 350 nm in diameterwere grouped together into one class. The obtained results showedthat all the casein micelle size distributions exhibited a maximum inthe diameter range of 100 to 150 nm and a minimum in the diameterlarger than 350 nm. Given that about 60% of the total volume occupiedby the casein micelles has a size of 100–200 nm, less than 18% of the vol-ume has a size of 50–100 nm and only less than 1% was larger than350 nm. However, from the control to the cryoconcentration cycle 3,the proportion of micelles with diameter of 100 to 150 nm increasedfrom 30.67% to 39.23%. The proportion of micelles smaller than 100 nmincreased also from 10.47% to 18.38%. In the class of micelle size of300–350 nm and N350 nm, the proportion of these micelles decreasedas the cryoconcentration cycle was increased. The reason of these chang-esmaybedue to themineral imbalance, especially Ca andMg ions,whichoccurred after the cryoconcentration fraction was separated (thawed).

The storage of the concentrates for up to 2 weeks at 4 °C had noeffect on the size distribution of casein micelles. The reason is likelythat the cryoconcentration process has no significant effect on the dis-tance between the casein micelles. However, after 3 weeks of storage,the distribution shifted toward larger micelles with average particlesize of 220 nm (Fig. 4). The increase in particle size from the 3rd weekstorage can be explained by the presence of a small number of largerparticles formed from micelle aggregation. Previously, large micelle

150–200 200–250 250–300 300–350

25.23 10.893 13.957 2.75322.412 9.057 10.87 2.00523.306 9.294 10.802 1.79422.353 8.607 9.614 1.454

Fig. 4. Particle size distributions after 3 weeks of storage: Unconcentrated skimmilk (C0),cryoconcentrated skimmilk at different cryoconcentration cycles: C1, C2, and C3: 1st, 2nd,and 3rd cryoconcentration cycle, respectively.

73A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

size indicating aggregation of casein micelles after 6 h has been ob-served by Liu et al. (2012), who studied the evaporative concentrationof skim milk and its effect on casein micelle hydration, composition,and distribution. In their work, the skimmilk samples were concentrat-ed at 50 °C in a rotary evaporator at 355± 5 Torr (0.47 bar) for differenttimes to acquire different concentrations of milk. Moreover, large mi-celle size was also observed after 8 h in the study of Bienvenue et al.(2003) on the rheological properties of concentrated skim milk as af-fected by the soluble minerals and the changes in its viscosity duringstorage. The increased micelle size observed in a previous study ofMartin, Williams, and Dunstan (2007) on skim milk powder may haveresulted from the duration it was kept under concentrated conditions.These results would suggest that the size of casein micelles is affectedby the contraction due to water removal and mineral imbalance.

3.5. Flow behavior

Fig. 5a shows the flow curves recorded at 25 °C of the control andskim milk samples cryoconcentrated from 9.25% to 25.12% total dry

Fig. 5. (a) Apparent viscosity of control (9.25%), cycle 1 (14.73%), cycle 2 (21.36%), and cycle 3 (week end 1 (S1), week end 2 (S2), week end 3 (S3), week end 4 (S4), week end 5 (S5).

matter. At this temperature, up to the 3rd cryoconcentration cycle, sim-ilarly to the control sample (initial skimmilk), all cryoconcentratedmilksamples exhibited a Newtonian behavior. Comparison between thecontrol (initial) milk and the samples obtained at the 1st and 2ndcryoconcentration cycles showed that the apparent viscosity decreasedslowly before to increase with increasing the shear rate. These resultsare in agreement with those reported by Sauer et al. (2012) on the ca-seins concentrated from2.5% up to 12.5%.However, for themilk concen-trate obtained at the 3rd cryoconcentration cycle, the apparent viscositydecreased slowly until about 25 s−1 with increasing the shear rate,which remained stable up to 100 s−1 and decreased rapidly 500 s−1.This behavior is called shear thinning and implies that the liquid hasnot true but an apparent viscosity. After 3 weeks of storage, the appar-ent viscosity of cryoconcentrated skim milk sample of 25.12% total drymatter (fraction of the 3rd cryoconcentration cycle) increased by nearly50% (Fig. 5b) and decreased rapidly with increasing shear rate, whichmay indicate a fragile (weak) inter-micellar links.

To accurately evaluate the most adapted flow behavior, variousmodels has been used in the literature to describe the dependence ofviscosity from shear rate: the power law model (Solanki & Rizvi,2001; Vélez-Ruiz and Barbosa-Cánovas, 1998), the Bingham model(Bienvenue, et al., 2003), and the Herschel–Bulkley model Vélez-Ruizand Barbosa-Cánovas (1998) are used. In our study, as the total drymatter content of the cryoconcentrated milk was b30%, the yield stresswas very small, and the viscosity data for all the cryoconcentratedmilk samples was fitted to Power law model and the values of κ and nwere thus determined. The fit for all data sets was very good with acoefficient of determination N98% in all cases. From time 0 to week 2,all cryoconcentrated milk samples exhibited a net Newtonian behavior(n very close to 1) for all the tested temperatures (5, 20, and 25 °C)(Fig. 6a), and from the week 3 to the week 4 of storage, the n indexdecreased with increasing concentration and decreasing workingtemperature. In the last week of storage (week 5), at all the tested tem-peratures, the obtained results showed a non-Newtonian behavior ofthe skim milk cryoconcentrated up to 25.12% dry matter (n b 1)(Fig. 6b). Previously, it has been showed that milk could be regardedas a Newtonian fluid when the total solids content of is ≤25% Changand Hartel (1997).

The flow parameters of the cryoconcentrated skim milk at 3 tem-peratures and 5 weeks of storage are presented in Tables 4 and 5.Temperature and concentration effects on the consistency coefficientwere well correlated by an exponential equation with a coefficient of

25.12%). (b) Apparent viscosity of factor 3 X as function of shear rate for week end 0 (S0),

Skim milk concentration (%)

Temperature (°C)Flo

w b

ehav

ior

inde

x

Week: 0, 1 and 2 a

b

Skim milk concentration (%) Tem

pera

ture

(°C

)

Flo

w b

ehav

ior

inde

x

Week: 5

Fig. 6. Combined effect of temperature and concentration on the flow behavior index weeks of storage: (a) weeks 0–2, (b) week 5.

74 A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

determination of a least 0.988. However, at the 5th week of stor-age, the combined effects of [concentration × temperature] and[concentration × week] on the consistency showed a highly significantdifference (P b 0.0001), and no significant difference (P = 0.0379) wasobserved for the combined effect of [temperature × week] on the con-sistency. Different results have been reported by Alvarez de Felipeet al. (1991) and Vélez-Ruiz and Barbosa-Cánovas (1998) Vélez-Ruizand Barbosa-Cánovas (1998) for sweetened condensed and evaporatedmilk, respectively, which can be explained by the structural build-up orgel formation developed during storage (Patil & Patel, 1992).

4. Conclusion

Skimmilkwas successfully cryoconcentrated by applying the cascadeeffect up to 25.12% total drymatter. After 3 cryoconcentration cycles, theprocess did not show any significant effect on the casein inter-micellardistance. Cryoconcentration resulted in a mineral imbalance, especiallyCa and Mg, and tended to reduce the size of the casein micelles as the

cryoconcentration cycle was increased. This reduction of the casein mi-celle size was reversible only after 3 weeks of refrigerated storage at4 °C. The flow behavior of the cryoconcentrated skim milk showed aNewtoniannature at all cryoconcentration cycles. The viscosity increasedwith the increasing the cryoconcentration cycle, but a low shear rate hadmuch more effect on the concentrated milk at higher cycles. After3 weeks of storage, the cryoconcentrated skim milk of 25.12% total drymatter showed a non-Newtonian flow behavior only at 5 °C, while atthe 5th week of storage, the cryoconcentrated milk with 25.12% totaldry matter content showed a non-Newtonian nature at all the testedtemperatures (5 °C, 15 °C, and 25 °C). The temperature had less effecton the viscosity versus concentration. Moreover, cryoconcentration ofskim milk significantly improved its color, as shown by its L* value,and yielded milk with a whiteness index similar to that of wholemilk. The results obtained on the effects of cryoconcentration onthe physico-chemical properties and rheological behavior duringstorage suggest that cryoconcentration cycles 1 and 2 would yieldbetter results for the use in the manufacture of dairy products.

Table 4Consistency index (K) of the cryoconcentrated skim milk at different temperatures and storage period (W1–5 = Weeks 1–5).

W1 W2 W3 W4 W5

K (mPa.sn) K (mPa·sn) K (mPa·sn) K (mPa·sn) K (mPa·sn)

C, % (w/w) 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C9.25 2.3 ± 0.01 1.5 ± 0.004 1.1 ± 0.01 2.5 ± 0.2 1.4 ± 0.11 1.1 ± 0.03 2.3 ± 0.007 2 ± 0.02 1.0 ± 0.08 2.40 ± 0.02 1.59 ± 0.02 1.1 ± 0.02 2.4 ± 0.03 1.3 ± 0.3 1.1 ± 0.04

14.73 4.7 ± 0.03 2.8 ± 0.11 1.9 ± 0.005 4.7 ± 0.01 2.8 ± 001 1.7 ± 0.01 5.1 ± 0.001 3 ± 0.06 1.8 ± 0.01 5.1 ± 0.23 4.1 ± 0.01 1.9 ± 0.17 5.2 ± 0.003 5.0 ± 0.001 2 ± 0.0321.36 12.3 ± 0.5 6.4 ± 0.3 4 ± 0.04 12 ± 0.07 6.3 ± 0.07 3.27 ± 0.02 13.4 ± 0.1 6 ± 0.08 3.6 ± 0.05 20.1 ± 0.7 9.8 ± 0.01 5.7 ± 0.05 21.9 ± 0.23 10.3 ± 0.1 5.9 ± 0.05

25.12 30.3 ± 0.3 13.8 ± 0.2 6.3 ± 0.2 30.9 ± 1.2 13.7 ± 0.07 6.7 ± 0.02 30.5 ± 1.6 13.9 ± 0.3 6.8 ± 0.03 41.5 ± 0.9 18.6 ± 0.1 10.6 ± 0.006 41.4 ± 0.4 21 ± 0.2 11.2 ± 0.23

Data are presented as mean ± standard deviation.

Table 5Flow behavior index (n) of the cryoconcentrated skim milk at different temperatures and storage period (W1–5 = Weeks 1–5).

W1 W2 W3 W4 W5

C, %(w/w)

n (dimensionless) n (dimensionless n (dimensionless n (dimensionless n (dimensionless

5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C 5 °C 15 °C 25 °C

9.25 1.012 ± 0.019 1.015 ± 0.004 1.015± 0.002 1.009 ± 0.001 1.01 ± 0.003 1.01 ± 0.011 1.012± 0.007 1.00 ± 0.001 1.005 ± 0.000 0.985 ± 0.027 1.00 ± 0.001 1.00 ± 0.000 0.909 ± 0.001 0.905 ± 0.01 0.905 ± 0.00514.73 1.00 ± 0.012 1.015 ± 0.000 1.021± 0.001 1.002 ± 0.002 1.009 ± 0.003 1.01 ± 0.001 1.005± 0.102 1.000± 0.000 1.01 ± 0.011 0.985 ± 0.029 0.992 ± 0.000 0.998 ± 0.011 0.894 ± 0.001 0.889 ± 0.000 0.898 ± 0.00321.36 1.002 ± 0.009 1.025 ± 0.001 1.011± 0.000 0.998 ± 0.001 1.001 ± 0.001 1.02 ± 0.000 0.999± 0.004 0.998± 0.002 1.005 ± 0.002 0.962 ± 0.031 0.992 ± 0.001 0.9755 ± 0.002 0.89 ± 0.002 0.89 ± 0.014 0.889 ± 0.00125.12 0.978 ± 0.003 0.987 ± 0.001 1.011± 0.001 0.985 ± 0.001 0.989 ± 0.001 0.988 ± 0.000 0.9 ± 0.002 0.989± 0.031 0.915 ± 0.001 0.908 ± 0.008 0.965 ± 0.032 0.965 ± 0.000 0.851 ± 0.000 0.868 ± 0.000 0.879 ± 0.001

75A.Balde,M

.Aider

/InnovativeFood

Scienceand

Emerging

Technologies34

(2016)68–76

76 A. Balde, M. Aider / Innovative Food Science and Emerging Technologies 34 (2016) 68–76

Acknowledgments

The authors express their entire gratitude to the Natural Sciencesand Engineering Research Council of Canada (NSERC) for the financialsupport. They heartily thanks Mrs. Diane Gagnon for the excellenttechnical support she provided during the realization of this work.

References

Aider, M., de Halleux, D., & Melnikova, I. (2009). Skim milk whey cryoconcentration andimpact on the composition of the concentrated and ice fractions. Food and BioprocessTechnology, 2(1), 80–88.

Aider, M., & Ounis, W. B. (2012). Skim milk cryoconcentration as affected by the thawingmode: Gravitational vs. microwave-assisted. International Journal of Food Science &Technology, 47(1), 195–202.

Alexander, M., Rojas-Ochoa, L., Leser, M., & Schurtenberger, P. (2002). Structure, dynam-ics, and optical properties of concentrated milk suspensions: An analogy to hard-sphere liquids. Journal of Colloid and Interface Science, 253(1), 35–46.

Alvarez de Felipe, A., Melcón, B., & Zapico, J. (1991). Structural changes in sweetened con-densed milk during storage: An electron microscopy study. Journal of Dairy Research,58(03), 337–344.

Anema, S. (2008). Effect of milk solids concentration on the gels formed by the acidifica-tion of heated pH-adjusted skim milk. Food Chemistry, 108(1), 110–118.

Bienvenue, A., Jimenez-Flores, R., & Singh, H. (2003). Rheological properties of concentrat-ed skim milk: Importance of soluble minerals in the changes in viscosity during stor-age. Journal of Dairy Science, 86(12), 3813–3821.

Carnovale, V., Britten, M., Couillard, C., & Bazinet, L. (2015). Impact of calcium on the in-teractions between epigallocatechin-3-gallate and β-lactoglobulin. Food ResearchInternational, 77(3), 565–571.

Chang, Y. -H., & Hartel, R. W. (1997). Flow properties of freeze-concentrated skim milk.Journal of Food Engineering, 31(3), 375–386.

Dahbi, L., Alexander, M., Trappe, V., Dhont, J., & Schurtenberger, P. (2010). Rheology andstructural arrest of casein suspensions. Journal of Colloid and Interface Science,342(2), 564–570.

De Kruif, C. (1999). Casein micelle interactions. International Dairy Journal, 9(3), 183–188.Di Cesare, L., Cortesi, P., & Martini, E. (1993). Studies on the concentration of model solu-

tions and fruit distillates by freezing-out'. Flüssiges Obst, 60(12), 442–445.Fellows, P. (2000). Food processing technology principles and practices (2nd (ed. ed.). Boca

Raton, FL: CRC Press.Flesland, O. (1995). Freeze concentration by layer crystallization. Drying Technology,

13(8–9), 1713–1739.Ghizzoni, C., Del Popolo, F., & Porretta, S. (1995). Quality of cryoconcentrated red fruit

juices as a function of volatile fraction. Rivista Italiana Eppos, 15, 5–21.Gunathilake, M., Dozen, M., Shimmura, K., & Miyawaki, O. (2014). An apparatus for partial

ice-melting to improve yield in progressive freeze-concentration. Journal of FoodEngineering..

Hwang, J., Lee, S., Park, H., Min, S., & Kwak, H. (2007). Comparison of physicochemical andsensory properties of freeze-concentrated milk with evaporated milk during storage.ASIAN AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES, 20(2), 273.

IDF, I. (2002). 185. Détermination de la teneur en azote. Méthode pratique par combus-tion selon le principe de Dumas. International Dairy Federation, Brussels, Belgium.

Iritani, E., Katagiri, N., Okada, K., Cao, D. -Q., & Kawasaki, K. (2013). Improvement of con-centration performance in shaking type of freeze concentration. Separation andPurification Technology, 120, 445–451.

Jeurnink, T. J., & De Kruif, K. G. (1993). Changes in milk on heating: Viscosity measure-ments. Journal of Dairy Research, 60(02), 139–150.

Karlsson, A. O., Ipsen, R., Schrader, K., & Ardö, Y. (2005). Relationship between physicalproperties of casein micelles and rheology of skim milk concentrate. Journal ofDairy Science, 88(11), 3784–3797.

Keshani, S., Luqman Chuah, A., Nourouzi, M., Russly, A., & Jamilah, B. (2010). Optimizationof concentration process on pomelo fruit juice using response surface methodology(RSM). Int Food Res J, 17, 733–742.

Liu, D. Z., Dunstan, D. E., & Martin, G. J. (2012). Evaporative concentration of skimmedmilk: Effect on casein micelle hydration, composition, and size. Food Chemistry,134(3), 1446–1452.

Martin, G., Williams, R., & Dunstan, D. (2007). Comparison of casein micelles in raw andreconstituted skim milk. Journal of Dairy Science, 90(10), 4543–4551.

Miyawaki, O., Liu, L., Shirai, Y., Sakashita, S., & Kagitani, K. (2005). Tubular ice system forscale-up of progressive freeze-concentration. Journal of Food Engineering, 69(1),107–113.

Mootse, H., Pisponen, A., Pajumägi, S., Polikarpus, A., Tatar, V., Sats, A., & Poikalainen, V.(2014). Investigation of casein micelle particle size distribution in raw milk ofEstonian Holstein dairy cows. Agronomy Research, 12(3), 153–158.

Morison, K., & Hartel, R. (2006). Evaporation and freeze concentration. Handbook of FoodEngineering (pp. 496–550).

Morris, G., Foster, T., & Harding, S. (2000). The effect of the degree of esterification on thehydrodynamic properties of citrus pectin. Food Hydrocolloids, 14(3), 227–235.

Owens, S., Brewer, J., & Rankin, S. (2001). Influence of bacterial cell population and pH onthe color of nonfat milk. LWT-Food Science and Technology, 34(5), 329–333.

Patil, G., & Patel, A. (1992). Viscoelastic properties of sweetened condensed milk as influ-enced by storage. Milchwissenschaft (Germany, FR).

Phillips, L. G., Mcgiff, M. L., Barbano, D. M., & Lawless, H. T. (1995). The influence of fat onthe sensory properties, viscosity, and color of lowfat milk. Journal of Dairy Science,78(6), 1258–1266.

Quiñones, H. J., Barbano, D. M., & Phillips, L. G. (1997). Influence of protein standardiza-tion by ultrafiltration on the viscosity, color, and sensory properties of skim and 1%milk. Journal of Dairy Science, 80(12), 3142–3151.

Sauer, A., Doehner, I., & Moraru, C. I. (2012). Steady shear rheological properties of micel-lar casein concentrates obtained by membrane filtration as a function of shear rate,concentration and temperature. Journal of Dairy Science, 95, 5569–5579.

Solanki, G., & Rizvi, S. (2001). Physico-chemical properties of skim milk retentates frommicrofiltration. Journal of Dairy Science, 84(11), 2381–2391.

Srilaorkul, S., Ozimek, L., Ooraikul, B., Hadziyev, D., & Wolfe, F. (1991). Effect of ultrafiltra-tion of skim milk on casein micelle size distribution in retentate. Journal of DairyScience, 74(1), 50–57.

Thijssen, H. (1970). Concentration processes for liquid foods containing volatile flavoursand aromas. International Journal of Food Science & Technology, 5(3), 211–229.

Thijssen, H., & Van Der Malen, B. (1981). Implications on quality of energy savings in theconcentration of foods. Resources and Conservation, 7, 287–299.

Vélez-Ruiz, J. F., & Barbosa-Cánovas, G. V. (1998). Rheological properties of concentratedmilk as a function of concentration, temperature and storage time. Journal of FoodEngineering, 35(2), 177–190.

Walstra, P. (1979). The voluminosity of bovine casein micelles and some of its implica-tions. Journal of Dairy Research, 46(02), 317–323.

Walstra, P., & Jenness, R. (1984). Dairy chemistry & physics. John Wiley & Sons.