Colloids and Surfaces B: Biointerfaces 126 (2015) 510519

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Colloids and Surfaces B: Biointerfaces

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Effect o /Nproper

Ben Aern HuDaniel V , Wa KU Leuven, Db KU Leuven, D iumc KU Leuven, D euven

a r t i c l

Article history:Received 2 SepReceived in re15 December 2014Accepted 4 January 2015Available online 10 January 2015

Keywords:Food emulsionMilkUltrasonic homVis/NIR spectrLight scatterinFat globule siz

roduisticsher re

development of new food products with improved properties. Visible and near-infrared (Vis/NIR) spec-troscopy is already widely used to measure the composition of agricultural and food products. However,this technology can also be consulted to acquire microstructure-related scattering properties of foodproducts. In this study, the effect of the fat globule size on the Vis/NIR bulk scattering properties of milkwas investigated. Variability in fat globule size distribution was created using ultrasonic homogenizationof raw milk. Reduction of the fat globule size resulted in a higher wavelength-dependency of both the

1. Introdu

Many prmayonnaisecolloids. Dition, distriborganolepticomplex iningredientsappearanceto measurefor better improve foimplementenable onli

CorresponE-mail ad

(B. Aernouts).

http://dx.doi.o0927-7765/ ogenizationoscopyge distribution

Vis/NIR bulk scattering coefcient and the scattering anisotropy factor. Moreover, the anisotropy factorand the bulk scattering coefcients for wavelengths above 600 nm were reduced and were dominatedby Rayleigh scattering. Additionally, the bulk scattering properties could be well (R2 0.990) estimatedfrom measured particle size distributions by consulting an algorithm based on the Mie solution. Futureresearch could aim at the inversion of this model to estimate the particle size distributions from Vis/NIRspectroscopic measurements.

2015 Elsevier B.V. All rights reserved.

ction

oducts in the food industry, including milk, ice cream,, jam, etc., are colloids or have been produced via

spersed particles determine, through their concentra-ution, size and structure, the physicochemical andc quality properties of the product. Additionally, theteractions among colloidal particles and with other

will contribute to the rheology, texture, stability,, and many other food characteristics [1]. Techniques

the particle properties of the colloid systems allowunderstanding these complex relations and furtherod processing techniques and recipes. Furthermore,ation of such sensing-technology in a food plant couldne monitoring of the colloids or the derived products

ding author. Tel.: +32 16321470.dresses: Ben.Aernouts@kuleuven.be, ben.aernouts@biw.kuleuven.be

during the production process and promote early detection of analtering product quality [2].

Milk is a natural colloidal emulsion of fat globules, as well asa hydrocolloid suspension of casein micelles, both dispersed ina water-based solution [3]. The fat globules, with a size rangingfrom 0.1 to 15 m, result from the assembly of small intracellularlipid droplets, followed by an apocrine secretion through the api-cal membrane of the epithelial cells in the mammary gland [4]. Thevolume fraction of these fat globules and casein micelles, the par-ticle size and shape and particleparticle interactions contributesignicantly to the quality properties of dairy products [5]. Amongthese inuential factors, the milk fat globule (MFG) size was foundto have an important impact on the physicochemical, functionaland sensory characteristics of the milk and derived products [59].The size of the native fat globules is correlated with the fatty acidcomposition and bioavailability of the lipids [1018]. Since the sizeof the globules, and more specically the amount of fat globulemembrane, has an impact on the enzymatic activity in the milk,it will affect the colloid stability. Furthermore, large fat globulescream faster and make skimming easier [13,19]. Centrifugation,

rg/10.1016/j.colsurfb.2015.01.0042015 Elsevier B.V. All rights reserved.f ultrasonic homogenization on the Visties of milk

outsa,, Robbe Van Beersa, Rodrigo Watta, Tjebbeermeulenc, Tom Van Gervenb, Jeroen Lammertyna

epartment of Biosystems, MeBioS, Kasteelpark Arenberg 30, 3001 Leuven, Belgiumepartment of Chemical Engineering, ProcESS, Willem de Croylaan 46, 3001 Leuven, Belgepartment of Biosystems, Livestock-Nutrition-Quality, Kasteelpark Arenberg 30, 3001 L

e i n f o

tember 2014vised form

a b s t r a c t

The size of colloidal particles in food pical, functional and sensory characterparticles could, therefore, help to furtIR bulk optical

ybrechtsa, Jeroen Jordensb,outer Saeysa

, Belgium

cts has a considerable impact on the products physicochem-. Measurement techniques to monitor the size of suspendedduce the variability in production processes and promote the

B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519 511

gravity separation and microltration are methods to separatenative MFGs of different sizes to create products with new, bet-ter and/or standardized properties [13,1922]. High-pressure orultrasonic homogenization can also reduce the size of the fat glob-ules by disthe MFG mwhey proteties for homMFGs [7,10,size of the fit is importity productThis is alsoMFG quantient cows, wquarters, anent times ofat content would allowproperties, This could rmote the deproperties athe size of Mcantly increcould give cow and ud

Optical mprocesses itroscopy stu(EMR) and penetrationible (Vis) anIt combinewith robustdays. OrigiwavelengthThe measurabsorb at tit can be cothe BeerLcontain locfrom its strto an unknmetal or memulsions; in biologicacessed foodthe predictmeasuremeence [32]. Mout of the tion will rethe scattersource of inangular- anical microsproperties aproperties ([6,2326,37

In the lasbased on Viabsorption ucts [3842is dened apath length

the need for (empirical) scatter corrections. The scattering, on theother hand, can be described with the bulk scattering coefcient s(cm1) and the angular scattering pattern: the normalized scatter-ing phase function p(). s describes the probability of scattering

it inled dn ofent atterients for bbetwd scaties f

the pese p

/NIR sheding tte thrderred latiolls ing patior Maand oays, te thse spcoune comerede algred beve

mayump

alsonditcrea

ase fe, ho

whreshon wm an

thiual stterisotrondenevalupropbustties the mect operts wi

BOP

s maencever, rupting them into smaller ones [2328]. However, asembranes are disrupted too and casein micelles andins adsorb at the increased surface area, the proper-ogenized MFGs are different compared to small native11,20]. Because of the impact of these techniques on theat globules and accordingly the food colloid properties,ant to monitor these processes to ensure a high qual-

with minimal intra- and inter-batch variability [6,29]. the case for raw milk as there is a large variability inty and size distribution, varying between milk of differ-ithin individual cows between different milkings andd within individual quarter milkings between differ-f sampling [6,2931]. Hence, online prediction of theand MFG size distribution, during the milking process,

for the separation of raw milk with different colloidaldepending on the needs of the food industry [32,33].educe the number of process steps in a food plant, pro-velopment of new food products with extra functionalnd upgrade the value of raw milk [29]. Additionally, asFG in milk from infected mammary glands is signi-

ased, MFG size information obtained on the dairy farminsight into the udder health status of each individualder quarter [34,35].easurement techniques are frequently used to monitor

n food industry and agriculture. Among them, spec-dies the interaction between electromagnetic radiationthe product of interest. Because of the relatively deep, relaxing the requirements for sample preparation, vis-d near-infrared (NIR) spectroscopy is often preferred.

s a fast and non-destructive measurement principle and cost-efcient sensor technology available nowa-nally, Vis/NIR spectroscopy was used to study the-dependent absorption by molecules in the product.ed absorption relates to the number of molecules thathat wavelength and the sample thickness. Therefore,nsulted to predict the products composition throughambert law. As most food and agricultural productsal non-uniformities, the radiation is forced to deviateaight trajectory and the travelling path is increasedown extent. These non-uniformities can be polymer,ineral particles in industrial systems; fat globules incells, cell organelles, air pores and brous structuresl tissues; or pores, air bubbles and crystals in pro-s, etc. These elastic scattering processes complicate

ion of the composition from measured spectra as thents have to be corrected for this non-linear interfer-oreover, samples with scattering properties that are

range compared to the samples used in the calibra-sult in inferior predictions [36]. On the other hand,ing properties could also be exploited as a valuableformation because the overall scattering level and thed wavelength-dependency are determined by the phys-tructure of the product. For emulsions, the scatteringre related to the quantity, size distribution and materialrefractive index) of the suspended spherical particles].t decade, several models and measurement techniquess/NIR spectroscopy have been proposed to quantify theand scattering properties for homogeneous food prod-]. The derived bulk absorption coefcient a (cm1)s the probability of absorption per unit innitesimal

and can be related to the product composition without

per una detaifunctiorepresthe scarepresfactor varies forwarpropertion offrom thfor Visestabliextendpromo

In omeasuplex reMaxwescatterthe eqution foinside Nowadsimuladisperinto action, thconsidof thesmeasu

Howwhich the assrithmsthis cowith ining phpracticinvalidtain thradiatimediuBeyondindivident scathe aniindepe

To tering and roproperthis is the effcal prosampleto thetions.

ThiconferMoreonitesimal path length, analogous to a, while p() givesescription of the normalized scattering probability as a

the scattering angle . As p() is often too complex tond interpret, only the rst moment of p(), dened asng anisotropy factor g, will generally be considered andthe mean cosine of the scattering angle. The anisotropyiological tissues and emulsions in the Vis/NIR rangeeen isotropic Rayleigh scattering (g = 0) and completettering (g = 1) [43]. Extraction of these bulk scatteringrom Vis/NIR spectroscopic measurements, and predic-article size distribution (PSD) of the measured emulsionroperties could create a whole new eld of applications

spectroscopy. As Vis/NIR spectrometers are already well for compositional analysis in the agro-food industry,heir functionalities with PSD estimations would furthere implementation of this technology [6,44].

to derive microstructure information (e.g. PSD) frombulk scattering properties (s and p()), the com-n between those two has to be unravelled by solvingequations for electromagnetic theory [43]. Since thearticles in an emulsion are almost perfectly spherical,ns can be evaluated with the Mie solution. The Mie solu-xwells equations describes the electromagnetic eldutside a spherical scatterer for given particle properties.algorithms that rely on the Mie solution are available toe bulk optical properties (a, s and p() or g) for poly-herical particles in an absorbing host medium, takingt the particles volume concentration and size distribu-plex refractive indices of particle and medium, and the

radiation wavelength [37,45]. Consequently, inversionorithms would clear the way for PSD estimations fromulk scattering properties [6].r, these algorithms build on a number of assumptions

not always be valid for food colloid systems. Apart fromtion of perfect sphericity of the particles, these algo-

assume that scattering events are independent. Underion, s follows a linear increase (through the origin)sing particle quantity, while the normalized scatter-unction and the anisotropy factor are independent. Inwever, the independent scattering relation becomes

en the particle volume concentration is above a cer-old. It has been shown that this limit depends on theavelength, refractive indices of the particles and hostd the particle size, shape and polydispersity [46,47].s limit, scattering particles are close enough such thatcattering events can inuence each other. This depend-ng typically reduces the bulk scattering coefcient andpy factor compared to what could be expected from thet scattering relations [4850].ate the concept of PSD estimation from the bulk scat-erties of milk, it is crucial to validate the accuracyness of the algorithms that relate the bulk scatteringto the particle size distribution of milk. Accordingly,ain objective of the present study. More specically,

f the fat globule size and quantity on the bulk opti-ies (BOPs) of milk was investigated by measuring milkth the golden standard method and comparing them

simulated from the measured particle size distribu-

nuscript is an extended and improved version of a proceeding presenting the preliminary results [40].the PSD measurements were measured with a laser

512 B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519

diffraction apparatus with improved sensitivity for submicronparticles (Section 2.2), while the accuracy of the unscatteredtransmittance measurements was further enhanced (Section2.3).

2. Materia

2.1. Milk sa

A fresh, rfarm. The mFT+ (Foss A9622:2000.respectivelysamples wiheated up into 7 subsand 68 mmtively 0 (rawwere generwith a CV23 mm micrmicrotip wpower leveto prevent homogenizthe milk saa constant uration of tefciency wmercial halmilk sampltent of respin this stud

The PSDall 8 samplpendent scathe milk satheir pure sdilution (1/

2.2. Milk padiffraction

The sizeenized mi(LD) after[711,141that the prsize distribu[28,59,62]. affect the abrane protebe determidissociationthere is (nesize.

The sizemined withglobules thmeasured wdynamic hythe actual particles anered with h

radius is signicantly larger than the particle-core radius [67]. Inthis study, however, the particle-core size is of interest as thisone relatesof the susp

is ret gene siz

]. As he sse sth ce

onlPSD quesf fat

mics si

sein hout,60,8his sts w

disn, U

spee re

(ISOeacho bren ratent

the fred aerice moerumtivelf t wal. Acht ict reonlyal of eata

ted bnd D

and3320elowe PS

PSDclearlobucase(

B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519 513

2.3. Measurement of bulk optical properties for raw andhomogenized milk samples

Double integrating sphere (DIS) and unscattered transmittancemeasurememine the Band were thand total tmilk samplof 600 m both equippmittance wdetector poin a similarbetween satered photo[48,88]. Thehigh signal-for very tursource (500high-precisment princmeasuremeple spectra10 nm by aples were ttemperatur

The invemized by Pthe diffuse measuremerefractive inand was caof signicatered transmfor raw miples homog550 nm [48

2.4. Simulahomogenize

The BOPwere also cto simulateticles in an solution forfor the effecing propertfractionalizis automatiprocedure itting procdation, the was used towavelengthindices obta

The BOPtion were ssignicantlyume concenprovided asfat globule casein fract(v/v), as dermilk densit

volume concentration was 2.00% (v/v), calculated from the crudeprotein content (Section 2.1), by taking into account the averagecasein fraction in crude protein (75.5%, w/w) and the density ofcasein (1.33 g/ml) and milk [87]. The real part of the refractive index

for milk fat, casein and milk serum was obtained by addingline to the Vis/NIR refractive index spectrum of water [90].seline was determined at 589 nm, a wavelength for whichractive indices are known for water, milk fat, casein and milk

[87,90]. The imaginary part of the refractive index for milkein and milk serum was calculated from the bulk absorp-efcients of respectively fat, protein and a mixture of water,

and serum proteins [37,9093]. The calculated BOP for then of fat globules and casein micelles were afterwards recom-similar to the recombination of different PSD fractions, to

the BOP of the milk samples [45].

ults and discussion

rticle size distribution of raw and homogenized milks

s were considered after they passed the quality inspectionsn Section 2.2. The nal cumulative volume-frequency PSDh of the 8 milk samples was calculated as the average of the

replicates and is presented for each sample in Fig. 1. For rawn exptwee

12 homng inmplger,s an

casebimoPSDsutiond innic h

miceed ea

the f

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Vol

ume

of p

artic

les s

mal

ler t

han

(*100

%)

he cumnized nts are considered to be the golden standard to deter-OP (a, s and g) for thin samples of turbid mediaerefore used in this study [88]. The diffuse reectanceransmittance were measured simultaneously on thee loaded in a borosilicate cuvette with a path lengthand positioned between the two integrating spheres,ed with a Vis and NIR detector. The unscattered trans-as measured in a separate path with the Vis and NIRsitioned 1.5 m behind the sample, which was loaded

cuvette with a path length of 170 m. A series of slitsmple and detector further reduced the number of scat-ns captured in the unscattered transmittance signal

sample illumination was especially designed to obtainto-noise spectra in the 5002250 nm wavelength rangebid media. It consists of a supercontinuum laser light2250 nm, 4 W optical power) in combination with aion monochromator. For more details on the measure-iple, the setup, its validation and the calibration andnt procedure, the reader is referred to [88]. All sam-

were measured from 500 until 1900 nm in steps ofutomated scanning of the monochromator. The sam-horoughly stirred before they were measured at roome.rse adding doubling (IAD) routine developed and opti-rahl [89] was consulted to obtain the BOP values fromreectance and total and unscattered transmittancents. Apart from the three measurements, also the realdex of the sample had to be provided to the algorithmlculated with the equation proposed in [87]. Becausent contribution of scattered photons to the unscat-ittance signals, no BOP estimation could be established

lk at wavelengths below 600 nm and for milk sam-enized for 20, 60, 120 and 300 s at wavelengths below,88].

tion of the bulk optical properties for raw andd milk samples

of the commercial, raw and homogenized milk samplesalculated from their respective PSD with an algorithm

the BOP of polydisperse suspensions of spherical par-absorbing host medium [45]. In this algorithm, the Mie

Maxwells equations has been generalized to accountt of an absorbing host medium on the particles scatter-ies. Polydispersity of the particle system is supported byation of the provided PSD, while the number of intervalscally optimized in an efcient iterative procedure. Thiss straightforward as it does not involve an inversion oredure. For more details on the algorithm and its vali-reader is referred to [45]. In this study, the algorithm

simulate the BOP of the samples in the 5001900 nm range, starting from the measured PSDs and refractiveined from literature.

for the fat globule fraction and casein micelle frac-imulated separately as their refractive indices differ. Moreover, the fat globule size distribution, fat vol-tration and fat and milk serum refractive indices were

inputs for the algorithm to simulate the BOP for thefraction, while a similar approach was followed for theion. The fat volume concentration was found to be 4.52%ived from the fat content (Section 2.1) and the fat andy of respectively 0.912 and 1.03 g/ml [87]. The casein

spectraa baseThe bathe refserumfat, castion colactosefractiobined, obtain

3. Res

3.1. Pasample

PSDgiven ifor eacpassedmilk, asize be0.8 andAs theresultimilk saand lonmicelle

Theof the sured distribresulteultrasocaseinassumsize of

Fig. 1. Thomogelicit bimodal PSD can be noticed. Casein micelles with an 0.01 and 0.8 m and fat globules with a size betweenm represent respectively the rst and second mode.ogenization time increases, fat globule size is reduced,

an overlap with the casein micelles. For the commerciale and for samples homogenized over a period of 300 s

no distinction in size can be made between the caseind fat globules.in micelle size distribution, derived from the rst modedal PSD for raw milk, was subtracted from all the mea-, resulting in the cumulative volume-frequency size

for the fat globules (Fig. 2). This subtraction method smooth monomodal PSDs, which indicates that theomogenization process had no noticeable effect on thelle volume fraction and size distribution [94], as wasrlier (Section 2.2). Fig. 2 clearly demonstrates that theat globules decreases with increasing homogenization

102 101 100 101

Diameter (m)

Raw20 s60 s120 s300 s600 s1200 sSkimCasein

ulative volume-frequency particle size distributions for the raw andmilk samples.

514 B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519

102 101 100 10 10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Diameter ( m)

Vol

ume

of p

artic

les s

mal

ler t

han

(*100

%)

Fig. 2. The cumulative volume-frequency particle size distributions for the raw andhomogenized fraction itself

time, whicderived for

The moFigs. 1 andDv50 and Dthe volumesented. Befhomogenizsmallest paerties reduchalf-skimmsize compaever, the PSFig. 2).

In respe(1250 maand 1200 s ultrasonic h1200 s of ulto be clearly

3.2. Measurhomogenize

The BOPthrough I

Fig. 3. Microscopic images (1250 magnication) of fat globules in raw milk. Theblack scale bar in the bottom-right corner represents 10 m.

icroscopic images (1250 magnication) of fat globules in the raw milk(Fig. 3) after 120 s of ultrasonic homogenization. The black scale bar in theright corner represents 10 m.

rements. For all the raw and homogenized milk sam-e measured bulk absorption coefcient spectra were very

R2 0.968) to each other and to the a spectrum of the water

Table 1Most importan(a) and after (brespectively thdiameter.

Sample

Raw 20 s 60 s 120 s 300 s 600 s 1200 s Skim Casein milk samples after subtraction of the casein contribution. The caseinis also illustrated.

h implicates that their quantity increases. The PSDthe casein fraction is also presented in Fig. 2.st important properties describing the PSDs in

2 are listed in Table 1. Next to the percentiles Dv10,v90, also the surface area moment mean (D32) and

moment mean diameter of the particles (D43) are pre-ore subtraction of the casein micelle PSD (Table 1(a)),ation had nearly no effect on the Dv10 as the 10% (v/v)rticles were mainly casein micelles. All other PSD prop-ed steadily with increasing homogenization time. Theed (Skim) milk sample contained fat globules of similarred to the milk sample homogenized for 1200 s. How-D width for the 1200 s sample is wider (Table 1(b) and

ctively Fig. 3, Fig. 4 and Fig. 5, the microscopic imagesgnication) of raw milk and milk homogenized for 120are illustrated. They clearly demonstrate the effect ofomogenization on the size of the fat globules. After

trasonic homogenization, the fat globules are too small noticeable with an optical microscope.

ement of the bulk optical properties for raw andd milk samples

Fig. 4. Msample bottom-

measuples, thclose (s for the 8 undiluted milk samples were obtainedAD from DIS and unscattered transmittance

fraction (89samples is eVis/NIR abs

t properties of the volume-frequency particle size distributions for the raw (raw), homo) subtraction of the casein micelle contribution. The properties of the casein micelle fracte 10th, 50th and 90th percentile of the volume-based particle size distribution. The D32

(a) With casein (m) (b) C

Dv10 Dv50 Dv90 D32 D43 Dv1

0.0306 0.411 5.69 0.0947 2.09 2.060.0295 0.312 4.67 0.0897 1.65 1.370.0288 0.222 3.30 0.0848 1.07 0.610.0288 0.195 2.35 0.0826 0.753 0.310.0288 0.165 1.27 0.0789 0.431 0.140.0285 0.143 0.777 0.0748 0.298 0.100.0287 0.127 0.518 0.0721 0.219 0.060.0248 0.0984 0.407 0.0606 0.167 0.07

0.02.7%, v/v) [90]. This is because the composition of thesexactly the same, and water is by far the most importantorbing milk component (Fig. 6). In respectively Fig. 7

genized (* s) and skimmed (skim) milk samples (Figs. 1 and 2) beforeion (casein) itself are also shown in (b). The Dv10, Dv50 and Dv90 are

and D43 are respectively the Sauter and DeBroukere mean particle

asein subtraction (m)

0 Dv50 Dv90 D32 D43

3.87 6.99 3.34 4.24 3.13 5.91 2.44 3.443 2.11 4.45 1.31 2.360 1.34 3.28 0.648 1.612 0.627 1.88 0.321 0.8441 0.389 1.22 0.226 0.54542 0.240 0.719 0.145 0.33653 0.253 0.655 0.162 0.31717 0.0724 0.264 0.0501 0.116

B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519 515

Fig. 5. Microscopic images (1250 magnication) of fat globules in the raw milksample (Fig. 3) after 1200 s of ultrasonic homogenization. The black scale bar in thebottom-right corner represents 10 m.

600 800 1000 1200 1400 1600 18000

5

10

15

20

25

30

35

40

Wavelength (nm)

a (cm

1 )

Raw20 s60 s120 s300 s600 s1200 sSkim

Fig. 6. Measured bulk absorption coefcient spectra (a) for the undiluted raw andhomogenized milk samples.

600 800 1000 1200 1400 1600 18000

100

200

300

400

500

600

Wavelength (nm)

s (cm

1 )

Raw20 s60 s120 s300 s600 s1200 sSkim

Fig. 7. Measured bulk scattering coefcient spectra (s) for the undiluted raw andhomogenized milk samples.

600 800 1000 1200 1400 1600 18000.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

Wavelength (nm)

Ani

sotro

py fa

ctor

g

Fig. 8. Measured scattering anisotropy spectra (g) for the undiluted raw and homog-enized milk samples.

and Fig. 8, the measured bulk scattering coefcient spectra (s) andscattering anisotropy spectra (g) are illustrated for the undilutedmilk samples. Both the s spectra (Fig. 7) and the g spectra (Fig. 8)are highly susceptible to the size (distribution) of the scattering fatparticles. Ultrasonic homogenization for less than 1 min resultedin an increaand a decrhomogenizVis/NIR ss spectra (), approafor the hal1200 s sammainly be consequent

The anVis/NIR raindependenglobule siz

0

100

200

300

400

500

600

700

800

s (cm

1 )

Fig. 9. Bulk scmilk samples tions (1/3) of tare rescaled toby respectivelse of the s for radiation wavelengths below 600 nmease of the s at longer wavelengths, while longeration induced an overall decrease of the samplesspectrum. Furthermore, with reducing particle size, thebecame more dependent on the radiation wavelengthching Rayleigh scattering (4) [43]. The s spectrumf-skimmed milk sample was lower compared to theple, despite the similar fat globule size. This can

explained by the lower fat volume concentration andly a lower quantity of scattering fat globules [2326].isotropy factor for the raw milk sample in thenge was relatively high (0.9340.960) and nearlyt of the radiation wavelength. With reducing fate, the anisotropy factor decreased and became more

Raw 1/3Raw 1/2Raw 1/1120 s 1/3600 800 1000 1200 1400 1600 1800Wavelength (nm)

120 s 1/2120 s 1/11200 s 1/31200 s 1/21200 s 1/1

attering coefcient s for raw and homogenized (for 120 s and 1200 s)measured on pure samples (1/1) and two- (1/2) and three-fold dilu-he same sample. The s spectra for the diluted samples (1/2 and 1/3)

the particle concentration of the pure sample through multiplicationy 2 and 3, following the linear independent scattering relation.

516 B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519

0

50

100

150

200

250 s

(cm

1 )

RawRaw*120 s

Fig. 10. Measthree-fold dilutering coefcie

wavelengthscattering wparticles [4establishedlengths abodiffuse reehigh absorpscattering (of each othequantity [4similar caseskimmed mBecause thethe casein mcontributedpared to 12globules (Ttrum and mspectrum fo

3.3. Effect ocoefcient in

To test tBOP for the 120 and 12after a two-water. The fold and thrand 2.17% (for the 3 pa3 samples (on the meaences causevolume contor derived is more proderived BOPthe anisotroconcentrati

In Fig. 9,and 1200 s)and 1/3) ar

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Ani

sotro

py fa

ctor

g

Measraw an

for t

ultie p

atter s sing. ing pcouldn (Fit in tampounds. Thp tilticleeseandenf at s, th3 saabsentrati

mulaenize600 800 1000 1200 1400 1600 1800Wavelength (nm)

120 s*1200 s1200 s*Casein*

ured and simulated (*) bulk scattering coefcient spectra (s) forted raw and homogenized milk samples. The simulated bulk scat-nt spectrum for the virtual casein fraction is also illustrated.

-dependent. This reduction in g indicates that the lightas more isotopic, which is typical for smaller scattering

3]. No accurate values for the anisotropy factor could be for the samples Skim and 1200 s for radiation wave-ve 1000 and 1450 nm, respectively. This was caused byctance values close to the noise level due to the verytion by water (89.7%, v/v) in combination with lows < 10 cm1). If scattering processes are independentr, the g spectrum should be independent of the particle8]. However, despite their similar fat globule size andin micelle size (Table 1(b)), the g spectrum for the half-ilk sample was lower compared to the 1200 s sample.

fat globule quantity was lower in sample Skim, whileicelle quantity was similar, the micelles would have

more to the nal g spectrum of sample Skim com-00 s. As the casein micelles were smaller than the fat

able 1(b) and Fig. 2), this resulted in a lower g spec-ore isotropic scattering (Fig. 11). Consequently, the gr sample Skim was lower.

f dependent scattering on the bulk scattering raw and homogenized milk samples

he validity of the independent scattering condition, the

Fig. 11. diluted spectrum

were mthe samdent scscaledscatterscattertra as relatiopresennized swere fsamplevalid uing parother rindepetions osampleand 1/likely concen

3.4. Sihomograw milk sample and the milk samples homogenized for00 s were, next to their pure state (1/1), also measuredfold (1/2) and three-fold (1/3) dilution with deionizedconcentration of scattering particles in the pure, two-ee-fold diluted samples was respectively 6.52%, 3.26%v/v). No consistent differences between the g spectrarticle concentrations could be detected for any of theresults not shown). This is probably because the noisesured g spectra was more signicant than the differ-d by the effect of dependent scattering at these particlecentrations. It is well known that an anisotropy fac-from DIS and unscattered transmittance measurementsne to measurement noise in comparison to the others [88]. Moreover, the effect of dependent scattering onpy factor is only signicant for relatively high volumeons of the scattering particles [48].

the measured s spectra for the 3 samples (raw, 120 s and the 3 considered particle concentrations (1/1, 1/2e shown. The s spectra for the 1/2 and 1/3 samples

As depeples and inthe simulatwere simulThe BOPs wand fat gloBOP of the mspectra maspectra areference bet(96.6%, v/v fsured spect(s) and scFig. 11. As between thfor the threment (R2 = 0g spectra f600 800 1000 1200 1400 1600 1800Wavelength (nm)

ured and simulated (*) scattering anisotropy spectra (g) for three-foldd homogenized milk samples. The simulated scattering anisotropyhe virtual casein fraction is also illustrated.

plied with respectively 2 and 3 to scale them all toarticle concentration according to the linear indepen-ing relation. Consequently, the differences between thepectra for the same sample are caused by dependentFor all samples, the highest volume concentration ofarticles (pure sample) clearly produced lower s spec-

be expected from the linear independent scatteringg. 9). Moreover, this effect was, to a smaller extent, stillhe s spectra for the two-fold dilution of the homoge-les. For the raw milk sample, no differences (R2 = 0.999)

between the scaled s spectra for the 1/2 and 1/3is indicates that the independent scattering relation isl higher particle volume concentrations if the scatter-s are larger and conrms the observations reported byrchers [48,88,9599]. Therefore, in raw milk samples,t scattering is valid up to particle volume concentra-least 3.26% (two-fold dilution). For the homogenizede difference between the scaled s spectra for the 1/2mples is very small. Therefore, dependent scattering ist in homogenized milk samples with particle volumeons up to 2.17% (three-fold dilution).

tion of the bulk optical properties in raw andd milk samplesndent scattering was present in the pure milk sam- most of the two-fold diluted samples (Fig. 9), whileion algorithm assumes independent scattering, the BOPated and validated for the three-fold diluted samples.ere simulated separately for the casein micelle fractionbules, and were recombined afterwards to obtain theilk samples. The simulated bulk absorption coefcient

tched well (R2 0.973) with the measured ones. These, however, not shown here as there was no visible dif-ween them and the a spectrum of their water fractionor three-fold diluted milk) [90]. The simulated and mea-ra for respectively the bulk scattering coefcient spectraattering anisotropy spectra (g) are shown in Fig. 10 andillustrated in these gures and Table 2, the agreemente simulated and measured bulk scattering propertiese-fold diluted samples is very good. The poorest agree-.777) was found between the simulated and measured

or the raw milk sample. As the anisotropy factor for

B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519 517

Table 2Coefcients of determination, indicating the agreement between measured and sim-ulated bulk optical properties for the raw and homogenized milk samples illustratedin Figs. 10 and 11.

Sample

Raw 120 s 1200 s Combined

raw milk isvery small a rather poosured bulk all three sambetween thindicated btively 0.990anisotropy measured asame. This inversion, tThe high cocal propertimeasuremealgorithm. Svalues in Fiment of theperfect sphever, they afor the refracomplex rethe entire Vsimulations

In contrpresent maulated and unscatteredity for subm

The simtering anisalso shownmicelles arlow and isMoreover, casein, relain a very the NIR. Amicelles wisamples. Limicelles wiand homog

Casein mnearly-sphespherical pawill be sufscattering preason, the might not bters could bHowever, aadditional soughly. As tof milk, it isrately. Ther

and without loss of micelles is essential in order to measure theirexact BOP and validate the simulated results.

To enable online PSD measurements on turbid colloids with ae volume concentration above 24%, dilution is not preferredce dependent scattering. Therefore, a model is needed thates tperting wsionis mo

case is, haveletry, sine msionscop

escrities relatnsfot the

clus

asonbulecattee consing d (NIredungthbsen(R2 d thverstion ties.

quaays, scopproartheent

wito enm blly, utioss, b

furthe Vi

wled

AerationRs2 Rg2

0.979 0.7770.990 0.9430.954 0.9640.990 0.996

nearly independent of the radiation wavelength, thedisagreement (RMSD = 2.33 103) already resulted inr R2. Comparison between all other simulated and mea-scattering properties resulted in an R2 above 0.94. For

ples together (raw, 120 s and 1200 s), the correlatione simulated and measured properties was very high,y the very high coefcients of determination (respec-

and 0.996) for the bulk scattering coefcients and thefactors (Table 2). The effect of homogenization on thend simulated bulk scattering properties is fairly theindicates the high potential of the Mie solution, aftero accurately estimate the PSD from BOP measurements.rrelation between simulated and measured bulk opti-es indicates a successful triple validation of (1) the PSDnts, (2) the BOP measurements and (3) the simulationmall differences between the simulated and measuredgs. 10 and 11 could be due to errors in the measure-

BOP and/or PSDs and/or because of invalidity of theericity and independent scattering assumptions. How-re more likely the result of incorrect simulation-inputsctive indices. Therefore, accurate measurement of thefractive indices for milk fat, casein and milk serum foris/NIR wavelength range could enable more accurate.ast to the preliminary results presented in [40], thenuscript shows a much better agreement between sim-measured BOP. Likely, this is the result of optimized

transmittance measurements and improved sensitiv-icron particles in the PSD measurements.ulated bulk scattering coefcient spectrum and scat-otropy spectrum for the virtual casein fraction are

in respectively Fig. 10 and Fig. 11. As the caseine relatively small (Table 1(b)), the g spectrum is

strongly dependent on the radiation wavelength.in combination with a low volume concentration oftive to the fat fraction (Section 2.4), this resultslow s spectrum, with nearly no scattering in

s a consequence, the s spectrum for the caseinll not contribute much to the s spectra of the milkkewise, also the simulated g spectrum for the caseinll have no substantial impact on the g spectra of the rawenized milk samples.icelles are, however, not perfectly spherical, but arical cluster of smaller casein sub-micelles [5]. For non-rticles, the simulated a and s for equivalent spheresciently accurate. However, the anisotropy factor g andhase function p() can differ signicantly [37]. For thissimulated g spectrum for the casein micelles in Fig. 11

particlto redudescribing proscattersuspenthat thand/ormodeltion wgeome

Onlsuspenspectrothat dpropertering the trapredic

4. Con

Ultrfat globulk sfore, bdecreainfrarefactor wavele

In arately size anfore, inestimaproperrelatednowadspectrothis apmilk, fudependneededorder tsize fro

Finacontriberthelewouldwith th

Ackno

BenFounde entirely correct. The T-matrix method for sphere clus-e a good alternative for the Mie solution [100102].

s it introduces extra degrees of freedom, it might importources of error and should, therefore, be validated thor-he fat globules dominate the bulk scattering properties

difcult to study the BOP of the casein micelles sepa-efore, isolation of the casein micelles without damaging

Robbe Van Bby the Instand Techno101552, 13acknowledgGlucoSens aand LA-110he effect of dependent scattering on the bulk scatter-ies. Recently, the PercusYevick model for dependentas proposed and successfully validated on colloidal

s [49,50,103,104]. Moreover, studies on milk showeddel is accurate for total volume fraction of fat globulesin micelles up to 45% [49,50]. Further exploration of thisowever, needed to evaluate the inuence of the radia-ngth, particle and host refractive indices and particleize, polydispersity and density [23,25].easurement of the BOP for an undiluted colloidal

is possible with time- or spatially-resolved Vis/NIRy [3842]. With the development of a general modelbes dependent scattering, measured bulk scatteringcould be transformed to follow the independent scat-ion. Consequently, after inversion of the Mie solution,rmed bulk scattering properties could be consulted toPSD of the suspended particles [105].

ions

ic homogenization of milk reduces the size of itss in relation to the exposure time. As a result, thering properties change signicantly and can, there-sulted to extract size distribution information. With

diameter of the fat globules, the visible (Vis) and near-R) bulk scattering coefcient and scattering anisotropyce and become more dependent on the radiation.ce of dependent scattering, the Mie solution can accu-

0.990) describe the relation between the fat globulee bulk scattering properties of a milk sample. There-ion of the Mie solution could allow for the accurateof the size distribution from measured bulk scatteringAs this would enable the prediction of microstructure-lity parameters, next to the compositional analysis usedthis would considerably increase the value of Vis/NIRy for agriculture and food industry. However, beforech can be executed on very turbid suspensions like purer research is required to develop a model that describesscattering in such media. Additionally, more research ish respect to the inversion of the Mie scattering model inable fast, accurate and robust prediction of the particleulk scattering properties.it was found that casein micelles only have a limitedn to the Vis/NIR bulk scattering properties of milk. Nev-etter understanding of the scattering by casein micelleser improve the model that related the fat globule size

s/NIR bulk scattering properties of the milk sample.

gements

nouts was funded as Ph.D. fellow of the Research-Flanders (FWO, grant 11A4813N). Rodrigo Watt,eers, Tjebbe Huybrechts and Jeroen Jordens are funded

itute for the Promotion of Innovation through Sciencelogy in Flanders (IWT-Flanders, respectively grants1777, 121611 and 121235). The authors gratefullye IWT-Flanders for the nancial support through thend Koesensor projects (respectively grants SB-090053770).

518 B. Aernouts et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 510519

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Effect of ultrasonic homogenization on the Vis/NIR bulk optical properties of milk1 Introduction2 Materials and methods2.1 Milk samples and ultrasonic homogenization of milk2.2 Milk particle size distribution measurements with laser diffraction2.3 Measurement of bulk optical properties for raw and homogenized milk samples2.4 Simulation of the bulk optical properties for raw and homogenized milk samples

3 Results and discussion3.1 Particle size distribution of raw and homogenized milk samples3.2 Measurement of the bulk optical properties for raw and homogenized milk samples3.3 Effect of dependent scattering on the bulk scattering coefficient in raw and homogenized milk samples3.4 Simulation of the bulk optical properties in raw and homogenized milk samples

4 ConclusionsAcknowledgementsReferences