TEXTURE ANALYSIS OF MILK PROTEIN GELS

USING DIGITAL IMAGE ANALYSIS

Juan Pablo Costa

1,2

, Horacio Castellini

, Patricia Risso

and Bibiana Riquelme

1,2

Dpto. de Química-Física, FCByF, Universidad Nacional de Rosario, Rosario, Argentina

Óptica Aplicada a la Biología, Instituto de Física Rosario (CONICET-UNR), Rosario, Argentina

Dpto. de Física, FCEIA, Universidad Nacional de Rosario, Rosario, Argentina

Keywords: Milk protein, Acid gel, Gel structure, Digital images, Glucono-delta-lactone, Bovine caseins.

Abstract: Sodium caseinate (NaCAS) is a very useful ingredient in food industry because of its nutritional and

functional properties. Acidification produces a gel structure as a result of the dissociation and aggregation

of caseinic fractions. Formation of these protein gels can be made by the slow reduction of pH through the

addition of glucono-delta-lactone (GDL). Depending on its concentration and temperature, hydrolysis speed

of GDL can affect the grade of hardness and elasticity of the formed gel. This study evaluated the effect on

the formation and structure of protein gels induced by different relations of GDL through analysis of digital

images obtained in an inverted conventional microscope and a confocal microscope. The entropy,

smoothness and variance decrease with the added GDL quantity, but the uniformity increases. Results

confirm that the texture depends on gelification speed, which is directly related to the amount of added

GDL. This digital image analysis technique using conventional or confocal microscopy is, therefore,

suitable and very useful for the texture analysis of acid gels formed by different GDL/NaCAS rates.

1 INTRODUCTION

The texture is a very important characteristic and its

analysis is a very useful tool to quantify and classify

objects or interest region in an image. Image texture

is a quantification of the space variation of

intensities that is impossible to define by its

sensorial character. There are several textural

parameters and algorithms proposed for the

quantification of an image texture such as the co-

occurrence matrix, statistical studies, the wavelet,

etc. (Jensen, 1996) All these techniques can be

useful to characterize a great variety of textures, but

they can be unsuccessful when the textures do not

show a periodic structure. A general assumption is

that the relevant information is in the space relation

inside the grayscale images.

Caseins (CN) represent the major protein

component of bovine milk. The CN precipitate at pH

4.6 and may be resolubilized by increasing the pH. If

the increase in the pH is carried out by the addition

of NaOH it is possible to end up obtaining sodium

caseinate (NaCAS). CN and NaCAS are extensively

used in food industry because of their

physicochemical, nutritional and functional

properties that make them valuable ingredients in

complex food preparations. Casein gels are

responsible for most of the rheological/textural

properties (i.e. stretch, fracture) of cheese and other

dairy products (Walstra, 1984; Mulvihill and Fox,

1989).

Dissociation and a further aggregation step of

CN fractions due to NaCAS acidification results in

the formation of a gel structure. A possible

explanation to this observation is that as pH is

adjusted towards the isoelectric point it causes a

decrease of the repulsive interactions, resulting in a

destabilization of the colloidal aggregates as pH

drops slightly below 5 at a given temperature (Braga

et al., 2006; Ruis et al., 2007). Nowadays, a process

that has gained the attention of food industry is

direct acidification by the addition of a lactone, such

as glucono-δ-lactone (GDL) which allows to

overcome some of the difficulties associated with

the traditional process of using bacteria. In fact, the

final pH of the system is a function of the amount of

GDL added whereas starter bacteria produce acid

until they inhibit their own growth as pH becomes

lower (Ruis et al., 2007; de Kruif, 1997).

322

Pablo Costa J., Castellini H., Risso P. and Riquelme B..

TEXTURE ANALYSIS OF MILK PROTEIN GELS USING DIGITAL IMAGE ANALYSIS .

DOI: 10.5220/0003289303220325

In Proceedings of the International Conference on Bioinformatics Models, Methods and Algorithms (BIOINFORMATICS-2011), pages 322-325

ISBN: 978-989-8425-36-2

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

Depending on GDL concentration and

temperature, hydrolysis speed of GDL can affect the

grade of hardness and elasticity of the formed gel.

The compactness and elasticity of gels formed at the

end of the acidification process of NaCAS depend

on the kinetic of the aggregation phenomena. As the

aggregation process becomes slower the more easily

a polypeptide chain could acquire different

orientations leading to the formation of more

compact gels with more elasticity and hardness

(Nespolo et al., 2010).

The interaction mechanisms and rheological

properties of obtained gels can be analyzed and

characterized by optical techniques (Yan et al.,

2008; Lucey, 2002).

In the present work, the gel structure formation

kinetics and different compaction degree were

evaluated by means of textural parameters from

digital image analysis. In order to do this, images of

bottom gel surface were obtained by conventional

inverted microscopy and images of gel internal

structure were obtained by confocal microscopy.

2 MATERIALS AND METHODS

2.1 Sample Preparation

In this work, the formation and structure of NaCAS

gels (3%) induced by different GDL/NaCAS ratio

(0.35; 0.5; 0.7 y 1.0) was evaluated by means of

analysis of digital images obtained in an inverted

optical and a confocal microscope.

An aqueous solution of bovine caseinate

(NaCAS) 3% w/w was prepared from the

commercial drug (Sigma Co.). The gelification

process was started adding GDL on 5 g of NaCAS

solution, at 35 ºC. The different amounts of GDL

corresponded to different relations GDL/NaCAS

(0.35, 0.5, 0.7 and 1) according to:

GDL

(1)

To obtain the microscopic images, 80 μL of each

sample at different R were placed in compartments

of the LAB-TEK II cells. The samples were made in

duplicate maintaining the incubation temperature at

35ºC.

2.2 Stained Protocol for Confocal

Microscopy

The gels were stained with Rhodamine B. In order to

do, a dilution 1/50 v/v of 1 mL of Rhodamine B at

0.01% in 49 mL of bidistilled water was prepared.

This medium was used to dissolve NaCAS to obtain

a solution at 3%.

2.3 Image Acquisition and Analysis

Transmission images of gels were obtained using a

conventional inverted microscopy (Union Optical)

with an objective 100x and a digital camera (Canon

PowershotA640) with a zoom 9.1x, for the different

GDL/NaCAS ratios.

Images of internal structure of gels stained with

Rhodamine B were obtained using a confocal

microscope (Nikon EZ-C1.) at (14.25 ± 0.05) µm on

the glass slide (inner the gel).

The effect on the formation and structure of

protein gels induced by different R was assessed

using analysis of microscopic digital images.

Previous to the analysis, the images were

transformed to numerical 8 bit formats RGB (Red,

Green, Blue) where each of them corresponds to

level color. All images were normalized to grayscale

by following transformation:

BGRY 114.0587.0299.0 +

(2)

Then, the values were normalized by mean of the

transformation:

minmax

min

)1(

−

−=

(3)

where L is the maximum gray level. It can see in

Figures 2 and 3.

In this numerical representation a simplest

statistical approaches were used. From normalized

gray level it obtained its histogram

()

, where

−

Li …

and L is the maximum gray level. As

well as texture estimations of obtained images, the

Shannon entropy S, smoothness R and uniformity U

were studied and they were defined by the following

equation (Gonzalez and Woods, 2002; Haralick,

1979):

()

∑

−

−=

))((log

NpNpS

(4)

)(1

−=

(5)

∑

−

)(

NpU

(6)

where

)(

is the variance. Because the

(

)

have values in the range form 0 to 1 and their sum is

TEXTURE ANALYSIS OF MILK PROTEIN GELS USING DIGITAL IMAGE ANALYSIS

323

equals 1, measure U is maximum for an image in

which all gray level are equal. Instead entropy is a

measure of variability and is 0 for a uniform image.

3 RESULTS AND DISCUSSIONS

Figure 1 shows the pictures registered at different

times after GDL addition for the visualization of

gelification kinetics.

Figure 2 and 3 show respectively the

transmission and confocal images of gels obtained

for R = 0.35, 0.5, 0.7 and 1.0. From these images, it

is possible to observe differences in the internal

microstructure of gels. These observations can be

quantified by the texture parameters as shown in

tables 1 and 2.

15 minutes 30 minutes

Figure 1: Visualization of gelification kinetics as a

function of GDL/NaCAS ratios (0.35, 0.5, 0.7 and 1.0).

(a) R = 0.35 (b) R = 0.5 (c) R = 0.7 (d) R = 1

Figure 2: Images of gels obtained using a conventional inverted microscopy (Union Optical) with an objective 100x and a

digital camera (Canon PowershotA640) with a zoom 9.1x, for the different GDL/NaCAS rates.

(a) R = 0.35 (b) R = 0.5 (c) R = 0.7 (d) R = 1

Figure 3: Images obtained by confocal microscopy inner the gels at (14.25 ± 0.05) µm on the slide.

Table 1: Texture parameters for the different GDL/NaCAS ratios. Digital images obtained by conventional inverted

microscopy of acid gels on the slides.

R Entropy Smoothness Variance Uniformity

0.35 7,68 ± 0,01 0,046 ± 0.001 3100 ± 100 0,0050 ± 0,0001

0.5 7,58 ± 0,01 0,034 ± 0.001 2300 ± 100 0,0058 ± 0,0001

0.7 7,47 ± 0,09 0,029 ± 0,004 2000 ± 200 0,0065 ± 0,0006

1 7,39 ± 0,03 0,026 ± 0,001 1750 ± 50 0,0069 ± 0,0002

Table 2: Texture parameters for the different GDL/NaCAS ratios. Digital images obtained by confocal microscopy of inner

structure of acid gel measured at 14 μm on glass slides.

R Entropy Smoothness Variance Uniformity

0.35 5.2 ± 0.1 0.012 ± 0.001 800 ± 90 0.038 ± 0.004

0.5 5.4 ± 0.2 0.014 ± 0.003 940 ± 100 0.030 ± 0.003

0.7 4.6 ± 0.1 0.014 ± 0.001 830 ± 100 0.051 ± 0.004

1 4.4 ± 0.1 0.015 ± 0.001 980 ± 90 0.056 ± 0.005

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4 CONCLUSIONS

Results show that the structure compaction, texture

and size of internal interstices depend on the

gelification rate, which is related to the GDL added

to the solution.

The entropy decrease and the uniformity

increases with the added GDL in the images

obtained using both microscopes, being the entropy

the parameter that has more high precision. Results

confirm that the texture depends on gelification

speed, which is directly related to the amount of

added GDL. Therefore, the present digital image

analysis technique is suitable and very useful to

characterize the texture of NaCAS acid gels formed

by different GDL ratios.

The present analysis technique of digital images

obtained by conventional and confocal microscopy

is suitable for a structural study of acid protein gels

and can be useful to evaluate the texture of dairy

products.

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