Effect of Acidification on Carrot (Daucus carota) Juice Cloud Stability

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Effect of Acidification on Carrot (Daucus carota) Juice Cloud Stability Alison K. Schultz, Diane Marie Barrett, and Stephanie R. Dungan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5042855 • Publication Date (Web): 29 Oct 2014 Downloaded from http://pubs.acs.org on November 6, 2014

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Journal of Agricultural and Food Chemistry

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Effect of Acidification on Carrot (Daucus carota) Juice Cloud Stability

Alison K. Schultza†, Diane M. Barretta and Stephanie R. Dungana,b* a

Department of Food Science and Technology, University of California, Davis, California 95616 b

Department of Chemical Engineering and Materials Science, University of California, Davis, California 95616

*Corresponding author [telephone (530) 752-5447; fax (530) 752-4759; email [email protected]]. †Current address: New Belgium Brewing, 500 Linden Street, Fort Collins, CO, 80524

ACS Paragon Plus Environment


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ABSTRACT Effects of acidity on cloud stability in pasteurized carrot juice were examined over the pH

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range of 3.5 to 6.2. Cloud sedimentation, particle diameter, and zeta potential were measured at

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each pH condition to quantify juice cloud stability and clarification during three days of storage.

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Acidification below pH 4.9 resulted in a less negative zeta potential, increased particle size and

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an unstable cloud leading to juice clarification. As the acidity increased, clarification occurred

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more rapidly and to a greater extent. Only a weak effect of ionic strength was observed when

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sodium salts were added to the juice, but addition of calcium salts significantly reduced the cloud

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stability.

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KEYWORDS: Daucus carota, carrot juice, acidification, clarification, zeta potential,

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electrostatic, flocculation, pectin


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INTRODUCTION

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The relatively high pH of carrot juice (pH~6) makes it unique among popular commercial

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juices, such as orange or apple juices which have pH values below 4.5. The low-acid nature of

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carrot juice may make it more susceptible to spoilage and pathogenic organisms, which can be

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countered by acidification. This issue is of particular interest in fresh carrot juice products that

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require refrigeration. In addition, carrot juice can be subjected to lower pH conditions when it is

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mixed with fruit juices in juice blend products. The purpose of this paper is to explore cloud

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stability in a low-acid juice like carrot juice, by examining the effect of pH on colloidal

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interactions between fine juice particles.

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Carrot juice, like all cloudy juices, is a two-phase colloidal system, with a dispersed solid

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phase called cloud, and a continuous liquid phase called serum.1 The serum contains water-

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soluble components, and the cloud is composed of insoluble particles of various sizes and shapes

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from the fruit tissue.1 Most juice clouds have average particle sizes between 0.2 and 10 µm and

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are frequently divided into coarse (> 10 µm) and fine (0.1-10 µm) cloud fractions.2-3 The

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accepted view of cloud particles is that they are a positive protein-carbohydrate complex

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surrounded by negatively-charged pectin, which gives the overall particle a negative charge.4-5

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Pectin is critically important in cloud stability, both because of its charge and its water-binding

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capabilities. When the pectin coat of particles binds with water, it forms a hydrated pectin layer

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that has been referred to as a hydration envelope; that is, water molecules are associating with

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the pectin, effectively enveloping the particle so that the density of the particle is reduced.6-7

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Reiter et al.3 analyzed the effect of acidification at various points during the production of

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juice from blanched carrots, using citric acid to acidify to pH 4.4. Using turbidity, particle size

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and charge measurements, the authors found that acidification at later stages of the production



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(after fine grinding or in the final juice) increased the particle size and reduced cloud stability.

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However, stability was enhanced when carrots were acidified earlier in the juice processing, an

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effect also observed by Sims et al.8 and Yu and Rupasinghe.9 The juice components may be

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affected by a decrease in pH in various ways. Proteins that are acid-sensitive may coagulate and

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precipitate when the pH is shifted from natural carrot pH (6.2) to 4.4.3 For pectin, its negative

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charge is pH dependent: pectic acid has a pKa of 3.5-4.0.6 Near the pKa pectin becomes

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substantially less negative and there is less repulsion between cloud particles.4,6 More broadly, as

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juice is acidified and pH is lowered, surface groups become increasingly protonated, which

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lowers the magnitude of the surface charge of the cloud particles. Reiter et al.3 concluded that

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the low pH caused particles to flocculate, but that the flocs could be broken up again or removed

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during processing if acidification occurred at earlier stages of the production.

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The current study was designed to investigate the underlying interparticle interactions

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affecting cloud stability in fresh carrot juice, through an analysis of particle size distributions and

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other cloud characteristics as a function of pH and ionic strength. Zeta potential measurements

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were employed in this study to characterize the electrical charge properties near the surface of

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the particles in suspension. An inverse relationship between zeta potential magnitude and

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aggregation has been shown to exist within the natural matrix of orange and apple juices.4,10-12

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There may be a relationship between the degree of methoxylation (DM) and the zeta potential of

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cloud particles. It was found that apple juice with a DM of 85-88% had a zeta potential of –19 to

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–20 mV,4,13 while orange juice with a DM of 71-73% had a more negative zeta potential of –23

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mV.4 For carrots (DM 55-69%), it is therefore predicted that the zeta potential would be more

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negative at the same pH than either that of apple or orange juice.14-17


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Acidification of juices is accomplished in industry either by combining a low-acid juice

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with a high-acid one, or by directly adding acid to the juice. A similar procedure was used to

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lower the pH of carrot juice in several of our experiments. However, such a procedure also alters

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the ionic strength and overall concentration of the juice, with the extent of these changes

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increasing with the degree of acidification. To control for this effect, in most experiments the

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juice pH was modified using citrate buffer salts at constant ionic strength. Given the possible role

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of double layer interactions between particles on cloud stability, juice ionic strength could well

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be an important parameter in clarification rates.

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Through research presented in this paper, we examine particle interactions as a function of

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pH, via static light scattering, turbidity and zeta potential, and we discuss the role of interparticle

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energy barriers in the observed cloud stability. We seek to elucidate cloud stability in low-acid

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carrot juice, which may have inherent properties different from those of juices previously studied

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that are naturally more acidic.

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MATERIALS AND METHODS

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Material sourcing

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Refrigerated fresh carrot juice ('ready to drink'), commercially produced in Bakersfield,

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California, was purchased from the local grocery store and used for all experiments. Juices

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prepared by us in the laboratory could not replicate the qualitative features and consistency of the

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particle size distributions of purchased fresh carrot juices, and thus a study on commercial juice

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was preferred. According to the manufacturer, the juice was produced from finely ground fresh

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carrots that were separated into juice and pomace; the juice was then pasteurized, packed, stored

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and distributed under refrigeration. There were no added ingredients and the juice was not



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acidified. As indicated by the manufacturer, the average total solids of the commercial juice was

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10-20% and the soluble solids was 5-6%. The weight fraction of all solids in carrot juice was

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estimated in our laboratory to be 0.09 g/g, by drying the juice in a 56˚C oven and weighing the

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residual. From this concentration was subtracted the amount of soluble solids in carrot juice, to

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give a value of ~0.04 g insoluble solids/g juice.18 Turbidity of the juice was measured in our

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laboratory using UV absorption at 660 nm to be 14cm–1. The range of pH for carrots used by the

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manufacturer was reported to be 5.85-6.45, and the pH of the unbuffered juice was measured by

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us to be 6.2.

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Citric acid monohydrate, sodium citrate, sodium chloride, and calcium chloride were

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obtained from Fisher Scientific (Fair Lawn, NJ).

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Acidification

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Citric acid and sodium citrate were added directly to commercial carrot juice to obtain seven

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samples of carrot juice with 0.1 M buffer concentrations and pH values spanning pH 3.5-6.1.

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Buffer salts were directly added to the carrot juice to ensure consistent ionic concentrations

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between the samples. All measurements were taken on the same day after three days of storage at

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7 °C to ensure particle flocculation was complete.

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Certain juice samples were acidified by adding various small volumes of 2 M citric acid solution to commercial carrot juice, in order to achieve the desired pH value.

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To explore the effect of neutralizing acidified juice back to its natural pH (6.2), 40 mL

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aliquots of juice were acidified with 2 M citric acid to pH 3.0 and left to sit for one day to allow

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flocculation to occur. The juice was then neutralized back to pH 6.2 using 2 M NaOH.


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Relative sediment height Relative sediment height, also known as the sedimentation test, is a measure of the quantity

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of visible settling that occurs in the juice.7,19 It is the height (Hsediment) of the cloud sediment

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divided by the total height (Htotal) of the juice in a vial.

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Roughly 5 mL of each juice sample was placed in a 7 mL glass vial with a screw cap and

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allowed to remain undisturbed except for gentle transportation of the vials from the refrigerator

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to the laboratory bench for measurements. The height of the liquid in the vials was between 25

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and 30 mm. A caliper was used to make height measurements in triplicate.

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Relative turbidity

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Relative turbidity is a spectrophotometric method to determine cloud stability by comparing

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the turbidity in a juice sample before and after centrifugation.3,5,7,12,20 For the carrot juices

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examined here, absorbance at 660 nm was used as the measure of juice turbidity. At this

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wavelength absorbance is predominantly determined by the scattering due to the particles, rather

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than specific chemical absorption.21 Relative turbidities were determined by measuring the

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absorbance values for the homogeneously mixed whole juice and for the supernatants obtained

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after centrifugation (4,200 × g, 15 min, 20ºC). When absorbance values measured in standard 1

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cm cuvettes were unacceptably high, shorter path length cuvettes (0.5 mm for whole juice and

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5.0 mm for juice supernatants) were used. The absorbance of the whole commercial juice was

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determined to be 0.68, and the supernatant was 0.81. Turbidity (τ) was calculated as the

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absorbance divided by the path length used for measurement. Relative turbidities (expressed as

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%) were calculated as τsuper/τtotal × 100, with τsuper obtained from the centrifuged supernatant layer

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and τtotal from the uncentrifuged whole juice. Measurements were performed in triplicate.



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Particle Size Determination Static light scattering was used to measure average particle size.3-5,7,11,22 Measurements

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were performed using a Microtrac S3500 (Montgomeryville, PA). Results were obtained as the

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volume fraction of particles contained within a discrete size range (bin). The mean diameter was

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determined as a volume average:

∑n d ∑n d

4 i i

D4,3 =

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i

3 i i

.

(1)

i

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Here D4,3 is the volume mean diameter, ni is the number of particles in bin i, and di is their

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€ gently ten times to ensure even distribution of particles and diameter. Carrot juice was inverted

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then added to the instrument sample port, which was operated at a flow rate of 15% of

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maximum. Particles were assumed to be irregular in shape and absorbing, and a refractive index

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value for water of 1.333 was used. Three replicates were measured for each sample.

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Zeta potential

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Using a Malvern Zetasizer (Westborough, PA), the zeta potential (ζ) of the cloud particles

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was determined by measuring the particles' electrophoretic mobility (µ) in the presence of an

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electric field. Carrot juice was diluted 1:40 with deionized water to ensure that it was in the

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appropriate concentration range for measurements, which in our experiments corresponded to a

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signal intensity of ~100 kcps. The dielectric constant and viscosity of the continuous phase were

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set to 78.3 F/m and 0.8903 mPa·s, respectively, at 25 ˚C. A minimum of ten analysis runs were

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performed for each sample, the values for which were averaged together and analyzed via

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Smoluchowski's equation, ζ = εµ/η, where ε is the dielectric constant and is η the viscosity of

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the continuous phase. This model requires the diffuse double layer to be thin compared to the

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particle radius. Three replicates were taken for each sample.


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RESULTS AND DISCUSSION

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Relative sediment height and relative turbidity

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Cloud stability decreased as juice pH was lowered by the use of citrate buffer. After three

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days of refrigerated storage there was almost no precipitate in the buffered control juice (pH 6.1),

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nor in the juices at the two next highest pH values, whereas a separate sediment layer was seen in

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more acidified juices (Figure 1). The extent of the separation was quantified by measuring the

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height of the sediment layer relative to the total juice height (Figure 2). This relative height

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increased with decreasing pH. These results were consistent with turbidity measurements of the

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supernatant before and after centrifugation (Figure 2). With a reduction in juice pH, the relative

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turbidity decreased monotonically, indicating that, most likely due to particle aggregation,

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particles sedimented more readily in lower pH samples upon centrifugation. The agreement

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between the results of relative sediment height and relative turbidity demonstrate that either

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method would be an appropriate way to measure the cloud instability in carrot juice. Results in

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Figure 1 also demonstrate that, as previously noted by Reiter et al.,5 formation of a sediment in

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this experiment was not accompanied by loss of visible turbidity in the supernatant, except to a

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small extent at the two lowest pH values.

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Particle size distributions

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Particle size distributions were measured for the unbuffered juice at pH 6.2 (Figure 3a), as

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well as the samples with added buffers (Figure 3b-h). The distribution for the control buffered

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juice (Figure 3b, pH 6.1), shows evidence of two overlapping modes: one with particle sizes of a

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few microns, and the other with submicron sizes. Particles of diameters less than 10 microns

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only were observed in the control juices, in contrast to typical juices analyzed by Reiter et al.,5



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which had particle sizes ranging from 0.1 to 700 µm. Thus, we did not observe a "coarse

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particle" fraction (defined as containing particles of diameter > 10 µm),5 and effects of

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acidification on fine particles (< 10 µm) could be monitored directly (Figure 3). The average

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particle size (D4,3) of the control juice (pH 6.1) was approximately 2 µm.

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Juices with buffer at high pH levels, i.e. close to the natural juice pH, had particle

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distributions that were similar to the control (Figure 3c). As pH decreased, the main peak shifted

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towards larger particle sizes and increased in height. These particle distributions also exhibited a

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shoulder to the left of the main peak, and this mode decreased in height as the pH decreased

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(Figures 3d-h). The rightward shift in the main peak with decreasing pH corresponded to only a

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moderate increase in the modal diameter (values marked in Figure 3), with this mode remaining

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below 10 µm for all pH conditions. Thus, it seems likely that upon acidification, cloud particles

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of various sizes (submicron and micron) flocculated together to form clusters with just a few

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members, resulting in fewer submicron particles in the cloud, and an accompanying growth in

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the size of the larger main mode. In addition, for pH values ≤ 4.6, a new mode developed at

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larger particle sizes above 10 µm (Figure 3f-h). The size of particles in this mode dramatically

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increased as the juice became more acidic. In contrast to particles in the main peak, it is clear that

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these largest particles are clusters with a large number of subparticles. These latter findings in

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particular are consistent with the work of Reiter et al.,3 who observed that acidification of carrot

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juice to pH 4.4 increased the average diameter of the particles above 10 µm.

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Overall, a monotonic increase in the average droplet size was observed with decreasing pH,

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as shown in Figure 4. Acidification caused particles to form aggregates that more rapidly

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sedimented and created an unstable cloud (Figures 1 and 2). Average particle size also increased

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in juices acidified by a method that was closer to industrial practice: by adding concentrated (2


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M) citric acid in increasing amounts to the original juice. Measured results for the average

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diameter in the entire juice under these conditions are shown in Figure 5a.

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Particle sizes in the supernatant and sediment of acidified (unbuffered) juices were also

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measured, with results in Figure 5a indicating that particles increased in size in both layers as the

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juices were acidified. During sedimentation of a monodisperse dispersion of particles, the

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vertical concentration profile exhibits three regions (Figure 5b): a stationary, concentrated region

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at the bottom of the container (the "sediment"); a region of intermediate concentration which still

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falls towards the sediment layer; and above that, a particle-free region. In a polydisperse mixture

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such as carrot juice cloud, these three distinct regions will occur simultaneously for different

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particle size fractions,23 as shown schematically for a bidisperse suspension in Figure 5c. The

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"supernatant" region observable above the sediment layer in Figure 1 therefore comprises a

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mixture of different particle sizes, in sedimenting layers progressing towards the bottom,

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stationary layer. The influence of pH on particle size in the supernatant is thus complex, as it

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results from opposing effects to the rate of particle settling and the size distribution remaining

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within the sedimenting supernatant regions. At high pH (pH≥5.0), little sedimentation occurred

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over the three days of the experiment, and therefore average sizes in the supernatant and

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sediment are the same as for the overall juice (Figure 5a). Under more acidic conditions,

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sedimentation partially separated the large and small particles, yielding smaller averages in the

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supernatant and larger in the sediment. However, because the sedimentation process was

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incomplete, the supernatant contained the still settling aggregates created at lower pH, and its

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average particle diameter is therefore larger than that measured at higher pH.

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The process of particle separation described above depends not only on the particle size, but

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on the strength of the forces exerted on the particles. Thus, we expect differences in the extent of



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particle separation in juices sedimenting due to gravity (Figures 1 and 5a), and juices subjected

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to centrifugation. After centrifugation for 15 min at 4200 × g, control and acidified carrot juices

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studied by Reiter et al.3 formed supernatants that displayed very similar particle size

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distributions. Large particles with diameters > ~5 µm that were present in the intact juices were

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mainly absent from the supernatant in all cases, leading the authors to conclude that acidification

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causes only coarse particles to aggregate.3 However, it is also possible that the centrifugation is

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effectively removing aggregates and other coarser particle fractions from all of the sample

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supernatants, regardless of the origins of those aggregates. In our results at lower pH values, we

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find that supernatants created by sedimentation of commercial carrot juice retained clear

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evidence of aggregates formed from fine particles.

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Aggregate formation and reversibility

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The effects of neutralizing acidified carrot juice back to its initial pH were also examined.

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Juice was acidified with 2 M citric acid to pH 3.0, causing the average particle size in the total

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juice to increase from 1.9 µm at the natural pH of carrot juice (6.2), to 13 µm at pH 3.0 (Figure

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5). This aggregate growth led to almost complete clarification upon centrifugation, with a

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relative turbidity of 5.2% (data not shown). The juice at pH 3.0 was then neutralized back to pH

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6.2 with addition of 2 M NaOH. One day after neutralization, the average particle size had

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decreased from 13 to 2.6 µm, and the relative turbidity increased to 14%, though it did not

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recover fully to the initial value of 39%.

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This partial recovery of cloud properties upon neutralization likely indicates that the

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aggregation of the particles at lower pH is reversible. These results are qualitatively similar to

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that seen in work done on the more acidic apple and orange juices. In these systems, adjusting

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the juices to pH 7 retarded clarification, and bringing the pH back down to the natural pH


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accelerated clarification.24-25 Ellerbee and Wicker11 also observed particle association and

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dissociation in orange juice aggregates at a fixed pH, which suggests formation of weak flocs.

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Likewise, carrot particles may flocculate at low pH, due to protonation and neutralization of the

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negatively-charged pectin groups at the particles’ surface, which reduces repulsion sufficiently to

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allow particles to be trapped together due to an attractive potential well, as discussed previously

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for orange juice.24 When base is added, and those groups are again deprotonated, the negative

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charges of the pectin become exposed again, and the particles repel one another. This apparently

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reverses flocculation, decreasing particle size. In this study, full recovery of the initial particle

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size may not have occurred because the attractive energy minimum between particles remained

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sufficiently large compared to thermal energy to allow for complete cluster separation. The

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protonation/deprotonation equilibrium of acid or base groups may also be altered within the

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cluster.

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The growth in particle size with acidification can be seen more directly by viewing the

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particles under a light microscope at 20× magnification (Figure 6). A substantial increase in

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particle size between pH 4.9 and pH 4.6 was particularly visible. At pH values above 4.6, the

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aggregates appeared to consist of small, rather symmetric clusters, whereas at the lower pH

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values the clusters appeared to become more irregular and elongated. In addition, the dispersions

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at the lowest pH displayed some very large aggregates, approaching 100 µm in size, consistent

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with results in Figures 3f-h.

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Zeta potential

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The zeta potential decreased in magnitude, from –31 mV to –15 mV, as pH decreased from

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6.1 to 3.9. Average particle size increased over this same range (Figure 4). Below pH 4.6,

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particle size increased and the magnitude of the potential decreased more steeply; this change



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corresponded to conditions where the large particle mode (diameter >10 µm) appeared and grew

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on the size distribution graphs (Figure 3) and in the micrographs (Figure 6). From these results, a

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clear correlation between zeta potential and particle size increase is apparent.

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Similar trends have been seen previously in apple and orange juices, which are naturally

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more acidic than carrot juice. Croak and Corredig4 found that, when they adjusted the pH of

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orange juice over the range of pH 2.5 to 6.0, the most negative zeta potential existed when the

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pH was 6.0, and its magnitude decreased steeply until it nearly reached 0 mV at pH 2.5. Ellerbee

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and Wicker11 also report a more negative zeta potential for pH 5.5 orange juice compared to that

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at the natural pH of 4.0. Larger average particle sizes and increased clarification were also

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observed at the lower pH condition. In apple juice, Benítez et al.26 measured zeta potential and

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turbidity in diafiltered apple juice after allowing one hour for sedimentation, comparing the

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control juice (pH 4.5) to those acidified with HCl to lower pH. Turbidity was the greatest, and

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thus the cloud most stable, at the highest pH of 4.5, where the zeta potential was also most

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negative. The zeta potential for carrot juice at pH 4 was found to be only slightly more negative

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(–17mV) than the value (–19mV) published for the fruit juices; this difference may reflect the

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lower DM for carrot pectin.

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For all three types of juice, decreasing the pH decreased the magnitude of the negative

325

charge on the particle, and this change likely reduced the energy barrier between the particles.

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Thus, particle flocculation and cloud instability was enhanced at lower pH in these three types of

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juice. Interestingly, Mensah-Wilson et al.7 demonstrated in a very different way the connection

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between the particle charge and cloud stability, by measuring the effects of adding pectin to

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pineapple juice and passion fruit nectar. The fruit particles were initially very weakly (positively)


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charged and would flocculate and separate, but became negatively charged and stabilized upon

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the addition of pectin.

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Acidification and ionic strength effects

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The practice of acidification by addition of different amounts of strong acid to the juice

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modifies both the concentration and ionic strength of the juice, in addition to its pH.

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Modification of pH by addition of 2 M citric acid required adding no more than 3% by volume of

336

the concentrated acid, and thus the dilution factor was insignificant. However, concentrations of

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the citric acid in the final juice ranged up to 0.062 M at the lowest pH, and this would increase

338

the juice ionic strength by up to ~0.04 M at pH values below 5.5.

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These acidified juices can then be compared to those in which buffer salts were added

340

directly in order to keep the ionic strength uniform between samples. The relative turbidity

341

results from Figure 2, in which citric acid/sodium citrate buffers were used to alter pH, are re-

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plotted and compared in Figure 7 with carrot juice to which 2 M citric acid was added to

343

decrease pH. Juices acidified using citrate buffer salts (prepared at a constant ionic strength of

344

0.1 M) showed a smoother increase in turbidity over the entire pH range, while the turbidity data

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of the juice acidified with 2 M citric acid displayed a more sigmoidal shape. The latter juices

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were slightly less stable at high and low pH than the juices containing buffer salts. This result is

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somewhat surprising, since the higher ionic strength in the buffer salt-containing mixtures might

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be expected to promote flocculation by decreasing the Debye length and lowering the energy

349

barrier between particles.10,13

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To probe this effect further, the extent of clarification of juices was compared when sodium

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chloride versus calcium chloride was added in specific amounts to unbuffered commercial carrot

352

juice at pH 6.2 (Figure 8). The juices were stored at 4 °C for six days, undisturbed, before



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relative sediment height was measured. The results in Figure 8 indicate that CaCl2 significantly

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induced clarification, in contrast to NaCl, which had little effect on the cloud stability. Results

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with the sodium salt are consistent with those in Figure 7, and indicate that any effect of Na+ on

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the particle diffuse double layer thicknesses does not seem to impact the particle stability.

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Benítez et al.10 explored the effect of ionic strength on apple juice cloud stability, by first

358

separating apple juice cloud particles from the juice serum and reconstituting them in aqueous

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solutions containing potassium chloride concentrations between 10–4 and 10–1 M. Turbidity, and

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hence cloud stability, was greatest at the lowest ionic strength, as expected.

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A key difference between the results in Figure 8 and those of Benítez et al.10 is that in the

362

latter study the contributions of the native juice serum have been removed. To account for the

363

surprisingly weak effect of ionic strength on our carrot juice cloud results, it is important to

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recognize that the native carrot juice serum will itself contain ions that contribute to the overall

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ionic strength. Data from the USDA National Nutrient database,27 as well as measurements of

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electrolyte concentrations in other types of juices28-32 suggest that native ionic strength values of

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the order of 20-100 mM10 are likely. At such concentrations, the diffuse double layer is already

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less than 1-2 nm, and further thinning upon addition of further ions may not have a large effect

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on modifying interparticle repulsion. In addition, with such small Debye lengths, electrostatic

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repulsion may occur over length scales comparable to short-range forces such as steric

371

interactions, giving the latter an important contributing role.33 Benítez et al.26 found that, even at

372

very low pH values where the zeta potential of isolated cloudy apple juice particles was close to

373

zero, particles remained significantly stable due to a short-range repulsion. Ackerley and

374

Wicker24 also concluded that at high pH values (pH 7), steric stabilization due to a hydrated

375

pectin layer accompanies repulsive forces in preventing aggregation and clarification of orange


Page 17 of 33


17 376

juice. Thus, despite the high pH and high negative charge in the native carrot juice, such short-

377

range interactions may be playing a significant role in carrot juice cloud stability, relative to the

378

longer range double layer interactions.

379

Results in Figure 8 also indicate an important contribution due to specific ion effects.

380

Previous research has demonstrated that the presence of divalent cations increases fruit juice

381

cloud instability, consistent with our results for carrot juice.11,34-36 Calcium bridging of pectin is

382

an important mechanism in the clarification of orange juice.37 Such particle destabilization has

383

been attributed to the formation of junction zones, in which divalent cations such as calcium

384

induce pectin chain association.34 Consequently, one explanation for the greater stability of the

385

juices modified with buffer salts (Figure 7), despite their higher ionic strength, is that the higher

386

concentration of sodium ions in the system competes with native calcium for binding to pectin.

387

This would make calcium-induced flocculation less likely in these samples compared to juices

388

acidified with citric acid.

389

pH Threshold for Carrot Cloud Instability

390

In summary, results from this study demonstrated that acidification of carrot juice caused

391

clarification of the cloud due to particle flocculation. The growth in particle size was attributed to

392

the particle zeta potential becoming less negative, which reduced the repulsion between particles

393

and allowed them to approach one another and aggregate. Effects on zeta potential and particle

394

clustering were observed over the entire pH of 3 to 6; however, the impact on cloud stability

395

became more marked for pH values below 5. At these lower pH values, particle clusters between

396

10 and 100µm developed as a significant portion of the size distribution. As the zeta potential

397

increases more strongly below pH 5, these larger clusters may be promoted by a reduction in the

398

energy barrier below a threshold value. However, the attractive interactions between particles in



Page 18 of 33

18 399

the cluster also appear quite weak, since the aggregation process was at least partially reversible

400

upon returning the juice to neutral pH.

401

However, salts had a complex effect on cloud stability. Small differences only were seen

402

between juices acidified with buffer salts at constant ionic strength, versus those acidified by the

403

addition of various amounts of concentrated citric acid; surprisingly, the juices at higher ionic

404

strength were slightly more stable. Addition of the monovalent sodium chloride to the juice had

405

no clear influence on the stability, whereas addition of divalent calcium chloride promoted

406

clarification, likely due to its ability to bind to pectin and form bridges between different pectin

407

molecules. Further work is needed to elucidate the types and concentrations of ions present in

408

carrot juice, in order to understand how these ions influence interparticle interactions. Factors

409

(such as chelators) that reduce the contribution of these ions in the juice, as well as factors

410

enhancing the negative charge of the particles, could push the threshold pH for carrot cloud

411

stability lower, and promote juice stability in the presence of acidification.

412 413

ABBREVIATIONS USED: DM (degree of methoxylation)


Page 19 of 33


19 414

REFERENCES

415

1. Castaldo, D.; Servillo, L.; Loiudice, R.; Balestrieri, C.; Laratta, B.; Quagliuolo, L.; Giovane,

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A. The detection of residual pectin methylesterase activity in pasteurized tomato juices. Int. J.

417

Food Sci. Tech. 1996, 31, 313-318.

418

2. Beveridge, T. Opalescent and cloudy fruit juices: formation and particle stability. Crit. Rev.

419

Food Sci. Nutr. 2002, 42, 317-337.

420

3. Reiter, M.; Stuparić, M.; Neidhart, S.; Carle, R. The role of process technology in carrot juice

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cloud stability. Lebensm.-Wiss. Technol. 2003, 36, 165-172.

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4. Croak, S.; Corredig, M. The role of pectin in orange juice stabilization: Effect of pectin

423

methylesterase and pectinase activity on the size of cloud particles. Food Hydrocolloids 2006,

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20, 961-965.

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5. Reiter, M.; Neidhart, S.; Carle, R. Sedimentation behaviour and turbidity of carrot juices in

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relation to the characteristics of their cloud particles. J. Sci. Food Agric. 2003, 83, 745-751.

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6. Carle, R. Cloud stability of pulp-containing tropical fruit nectars. Fruit Process. 1998, 8,

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266-272.

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7. Mensah-Wilson, M.; Reiter, M.; Bail, R.; Neidhart, S.; Carle, R. Cloud stabilizing potential

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of pectin on pulp-containing fruit beverages. Fruit Process. 2000, 2, 47-54.

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8. Sims, C. A.; Balaban, M. O.; Matthews, R. F. Optimization of carrot juice color and cloud

432

stability. J. Food Sci. 1993, 58, 1129-1131.

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9. Yu, L. J.; Rupasinghe, H. P. V. Effect of acidification on quality and shelf-life of carrot juice.

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Can. J. Plant Sci. 2012, 92, 1113-1120.

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10. Benítez, E. I.; Lozano, J. E.; Genovese, D. B. Fractal dimension and mechanism of

436

aggregation of apple juice particles. Food Sci. Technol. Int. 2010, 16, 179-186.



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11. Ellerbee, L.; Wicker, L. Calcium and pH influence on orange juice cloud stability. J. Sci.

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Food Agric. 2011, 91, 171-7.

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12. Mollov, P.; Mihalev, K.; Buleva, M.; Petkanchin, I. Cloud stability of apple juices in relation

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to their particle charge properties studied by electro-optics. Food Res. Int. 2006, 39, 519-524.

441

13. Genovese, D. B.; Lozano, J. E. The effect of hydrocolloids on the stability and viscosity of

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cloudy apple juices. Food Hydrocolloids 2001, 15, 1-7.

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14. De Roeck, A.; Mols, J.; Duvetter, T.; Van Loey, A.; Hendrickx, M. Carrot texture

444

degradation kinetics and pectin changes during thermal versus high-pressure/high-temperature

445

processing: A comparative study. Food Chem. 2010, 120, 1104-1112.

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15. Ng, A.; Waldron, K. W. Effect of cooking and pre-cooking on cell-wall chemistry in relation

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to firmness of carrot tissues. J. Sci. Food Agric. 1997, 73, 503-512.

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16. Smout, C.; Sila, D. N.; Vu, T. S.; Van Loey, A. M. L.; Hendrickx, M. E. G. Effect of

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preheating and calcium pre-treatment on pectin structure and thermal texture degradation: a case

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study on carrots. J. Food Eng. 2005, 67, 419-425.

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17. Trejo Araya, X. I.; Hendrickx, M.; Verlinden, B. E.; Van Buggenhout, S.; Smale, N. J.;

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Stewart, C.; John Mawson, A. Understanding texture changes of high pressure processed fresh

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carrots: A microstructural and biochemical approach. J. Food Eng. 2007, 80, 873-884.

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18. Demir, N.; Bahceci, K. S.; Acar, J. The effect of processing method on the characteristics of

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carrot juice. J. Food Qual. 2007, 30, 813-822.

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19. Silva, V. M.; Sato, A. C. K.; Barbosa, G.; Dacanal, G.; Ciro-Velásquez, H. J.; Cunha, R. L.

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The effect of homogenisation on the stability of pineapple pulp. Int. J. Food Sci. Tech. 2010, 45,

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2127-2133.


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20. Ibrahim, G. E.; Hassan, I. M.; Abd-Elrashid, A. M.; El-Massry, K. F.; Eh-Ghorab, A. H.;

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Manal, M. R.; Osman, F. Effect of clouding agents on the quality of apple juice during storage.

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Food Hydrocolloids 2011, 25, 91-97.

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21. Harrison, D.; Fisch, M. Turbidity Measurement. In Measurement, Instrumentation, and

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Sensors Handbook, 2nd Edition [Online]; Webster, J.G . and Eren, H., Eds.; CRC Press LLC:

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2014; ch 51, pp 1-10. http://dx.doi.org/10.1201/b15664-57 (accessed 05/01/2014).

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22. Wicker, L.; Ackerley, J. L.; Corredig, M. Clarification of juice by thermolabile Valencia

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pectinmethylesterase is accelerated by cations. J. Agric. Food Chem. 2002, 50, 4091-4095.

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23. Bürger, R.; Concha, F.; Fjelde, K. K.; Karlsen, K. H. Numerical simulation of the settling of

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polydisperse suspensions of spheres. Powder Technol. 2000, 113, 30-54.

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24. Ackerley, J.; Wicker, L. Floc formation and changes in serum soluble cloud components of

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fresh Valencia orange juice. J. Food Sci. 2003, 68, 1169-1174.

471

25. Yamasaki, M.; Kato, A.; Chu, S.-Y.; Arima, K. E. I. Pectic enzymes in the clarification of

472

apple juice. II The mechanism of clarification. Agric. Biol. Chem. 1967, 31, 552-560.

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26. Benítez, E. I.; Genovese, D. B.; Lozano, J. E. Effect of pH and ionic strength on apple juice

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turbidity: Application of the extended DLVO theory. Food Hydrocolloids 2007, 21, 100-109.

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27. Holden, J. National Nutrient Database for Standard Reference. USDA, Ed.: National

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Agricultural Library, 2012.

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28. Eisele, T. A.; Drake, S. R. The partial compositional characteristics of apple juice from 175

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apple varieties. J. Food Compos. Anal. 2005, 18, 213-221.

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29. Fung, Y. S.; Lau, K. M. Separation and determination of cations in beverage products by

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capillary zone electrophoresis. J. Chromatogr. 2006, 1118, 144-150.



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30. Genovese, D. B.; Lozano, J. E. Particle size determination of food suspensions: Application

482

to cloudy apple juice. J. Food Process Eng. 2000, 23, 437-452.

483

31. Tormen, L.; Torres, D. P.; Dittert, I. M.; Araújo, R. G. O.; Frescura, V. L. A.; Curtius, A. J.

484

Rapid assessment of metal contamination in commercial fruit juices by inductively coupled mass

485

spectrometry after a simple dilution. J. Food Compos. Anal. 2011, 24, 95-102.

486

32. Vera, E.; Sandeaux, J.; Persin, F.; Pourcelly, G.; Dornier, M.; Piombo, G.; Ruales, J.

487

Deacidification of clarified tropical fruit juices by electrodialysis. Part II. Characteristics of the

488

deacidified juices. J. Food Eng. 2007, 78, 1439-1445.

489

33. Genovese, D. B.; Lozano, J. E. Contribution of colloidal forces to the viscosity and stability

490

of cloudy apple juice. Food Hydrocolloids 2006, 20, 767-773.

491

34. Braccini, I.; Perez, S. Molecular basis of Ca2+-induced gelation in alginates and pectins: the

492

egg-box model revisited. Biomacromolecules 2001, 2, 1089-96.

493

35. Dronnet, V. M.; Renard, C. M. G. C.; Axelos, M. A. V.; Thibault, J. F. Characterisation and

494

selectivity of divalent metal ions binding by citrus and sugar-beet pectins. Carbohydr. Polym.

495

1996, 30, 253-263.

496

36. Whitaker, J. R. Pectic substances, pectic enzymes and haze formation in fruit juices. Enzyme

497

Microb. Technol. 1984, 6, 341-349.

498

37. Corredig, M.; Kerr, W.; Wicker, L. Particle size distribution of orange juice cloud after

499

addition of sensitized pectin. J. Agric. Food Chem. 2001, 49, 2523-6.

500 501 502

FUNDING: The authors would like to thank the NSF Center for Advanced Processing and

503

Packaging Studies for their funding of this research.


Page 23 of 33


23 504

FIGURE CAPTIONS

505

Figure 1. Cloud sedimentation in buffered commercial carrot juice after 3 days in refrigerated

506

storage. pH 6.1 is the control buffered juice.

507

Figure 2. Relative turbidity (τsuper/τtotal, ) and relative sediment height (Hsediment/Htotal, ☐) as a

508

function of pH of commercial carrot juice with buffer salts added.

509

Figure 3. Particle size distribution graphs showing the volume fraction with the associated

510

particle diameter (±0.04 µm) of (a) control unbuffered juice (pH 6.2), and juices with added

511

buffer salts: (b) pH 6.1, (c) pH 5.8, (d) pH 5.4, (e) pH 4.9, (f) 4.6, (g) pH 4.2, and (h) pH 3.9.

512

Particle diameter at the maximum volume fraction is indicated above the main peak.

513

Figure 4. Average particle size D4,3 () and zeta potential (☐) of commercial carrot juice

514

samples with added buffer.

515

Figure 5. Effect of gravitational separation and pH on particle size. (a) D4,3 measured in the

516

entire juice (black bar), in the sediment (gray bar) and in the supernatant (white bar) in

517

commercial juice acidified with 2 M citric acid and allowed to separate for 3 days; (b) particle

518

layers in a monodisperse suspension during settling; (c) particle layers in a bidisperse suspension

519

during settling.

520

Figure 6. Optical micrographs with 20× magnification of carrot juice cloud particles at different

521

pH values created by acidification of the juice.

522

Figure 7. Relative turbidity (τsuper/τtotal) as a function of pH of commercial carrot juice with 2M

523

citric acid solution () or buffer salts (☐) added. Buffer salts contributed 0.1 M to the ionic

524

strength at all pH values.



Page 24 of 33

24 525

Figure 8. Relative sediment height (Hsediment/Htotal) in commercial carrot juice (no acidification)

526

after 3 days storage at 4˚C, with added CaCl2 (☐) or NaCl ().


Page 25 of 33


25

Figure 1. pH 3.9 pH 4.2

pH 4.6

pH 4.9

pH 5.4

pH 5.8

pH 6.1



Page 26 of 33

26

Figure 2. 14 10

25 20

8

Relative turbidity

6

15 10

4

5

2 0

0 3.5

Hsediment/Htotal (%)

12

τsuper/τtotal (%)

30

Relative sediment height

4.0

4.5

5.0

pH

5.5

6.0

6.5


Page 27 of 33


27

Volume Fraction (%)

(a) pH 6.2

80 70 60 50 40 30 20 10 0

2.0 µm

0.1

1

10

Volume Fraction (%)

Volume Fraction (%)

0

40 20 0 1

10

0 1

10

60 40 20 0 0.1

1

10

Diameter (µm)

20 0 1

80

100

10

4.2 µm

100

(f) pH 4.6

60 40 20 0 0.1

1

10

100

Diameter (µm)

(g) pH 4.2 5.0 µm

80

40

100

Diameter (µm)

(d) pH 5.4

Diameter (µm)

Volume Fraction (%)

20

100

60

40

10

2.5 µm

0.1

3.6 µm

0.1

80

100

(e) pH 4.9

80 60

1

Diameter (µm)

2.0 µm

Volume Fraction (%)

20

(c) pH 5.8

Diameter (µm)

Volume Fraction (%)

40

80

0.1

1.8 µm

60

0.1

Size (µm)

60

(b) pH 6.1

80

100

Volume Fraction (%)

Volume Fraction (%)

Figure 3.

80

6.0 µm

(h) pH 3.9

60 40 20 0 0.1

1

10

Diameter (µm)


100


Page 28 of 33

28

9 8 7 6 5 4 3 2 1 0

0 -5 Diameter

-10 -15 -20

Zeta potential

-25 -30

Zeta Potential (mV)

Average diameter (µm)

Figure 4.

-35 3.5

4.0

4.5

5.0 pH

5.5

6.0

6.5


Page 29 of 33


29

Figure 5.

Average diameter (µm)

(b) (a)

(c)

Total Juice Sediment Supernatant

pH

5.5

6.0

6.2

5.0

4.5

4.0

3.5

3.0

18 16 14 12 10 8 6 4 2 0



Page 30 of 33

30

Figure 6.

pH 3.9

pH 4.2

pH 4.6

pH 4.9

pH 5.4

pH 5.8

pH 6.1


Page 31 of 33


31

Figure 7. 14

citrate buffer

τsuper/τtotal (%)

12 10 8 citric acid

6 4 2 0 3.5

4

4.5

5

5.5

6

6.5

pH



Page 32 of 33

32

Figure 8. CaCl2

Hsediment/Htotal (%)

20 15 10

NaCl

5 0 0

20

40

60

80

100

Salt Concentration (mM)


Page 33 of 33


33 For Table of Contents Only


Effect of Acidification on Carrot (Daucus carota) Juice Cloud Stability

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