Rheological properties and Microstructure of Acid Milk Curd

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Rheological Properties and microstructure of Acid Milk Curd by Curdlan Addition, a Polysaccharide from Bacteria

(Sifat-Sifat Reologi dan Mikrostruktur Curd Susu Asam dengan Penambahan Curdlan, Polisakarida Bakteri)

Ratmawati Malaka1 and Sudirman Baco¹

Abstrak

Dalam industri susu, penggunaan polisakarida yang diproduksi bakteri adalah penting dalam peningkatan bentuk dan tekstur produk akhir. Curdlan ditambahkan dalam makanan untuk meningkatkan kualitasnya dan juga digunakan untuk membuat makanan baru. Tujuan penelitian ini adalah untuk mengetahui sifat-sifat reologi dan mikrostruktur curd susu asam dengan penambahan curdlan yang difermentasi dengan bakteri asam laktat. Curd susu asam dibuat dari susu skim rekonstitusi 10%, ditambahkan dengan 0 – 1% curdlan, kemudian dipanaskan pada suhu 85^oC selama 30 detik, diinokulasi dengan 1% Lb. delbrueckii subsp. bulgaricus B-5b dan diinkubasi pada 37^oC selama 16 jam. Viskositas, pH, hardness dan breaking energy curd susu asam yang mengandung curdlan meningkat dengan meningkatnya konsentrasi curdlan. Pada kontrol curd susu asam tanpa curdlan hanya kasein misel dan Lb. delbrueckii subsp. bulgaricus B-5b yang terdapat dalam mikrostruktur gel. Secara umum, sedikit meningkat derajat ikatan kasein misel menjadi rantai dan kluster dengan adanya curdlan dibandingkan dengan curd susu asam kontrol. Curdlan saat berinteraksi dengan kasein misel, kelihatannya seperti massa benang halus yang bergabung seperti tali antara satu kasein dengan kasein lainnya, dan/atau sebagai lapisan massa benang halus pada permukaan kluster kasein misel.

Kata Kunci: Polisakarida Bakteri, Curdlan, Curd Susu Asam, Sifat-Sifat Reologi, Mikrostruktur.

Abstract

In dairy industry, the use of polysaccharide producing bacteria is of interest with respect to improvement of body and texture of milk product. Curdlan is added to foods to improve their properties and is also used to make new foods. The objective of this study was to investigate the rheological properties and microstructure of acid milk curd with curdlan addition, fermented by lactic acid bacteria. The acid milk curd was made from 10% reconstituted skim milk (RSM), added with 0 - 1% of curdlan, heated at 85ºC for 30 sec, inoculated with 1% of Lb. delbrueckii subsp. bulgaricus B-5b, and incubated at 37ºC for 16 h. The viscosity, hardness and breaking energy of acid milk curd containing curdlan increased with increasing curdlan concentration. In a control acid milk curd containing no curdlan (0% of curdlan), only casein micelles and Lb. Delbrueckii subsp. bulgaricus B-5b composed the gel microstructure. In general, there was somewhat higher degree of linking casein micelles into chains and clusters in the presence of curdlan than in control acid milk curd. When curdlan interacted with casein micelles they appeared as fluffy mass joined by string between one-casein and the other, and/or a fluffy mass film on the surface of clusters of casein micelles.

Key word : Microbial polysaccharide curdlan, acid milk curd, rheological properties, Microstructure.

Introduction

There have been many investigations involving optimization of milk curd texture. These studies have demonstrated that the total solid and fat levels in the milk, heat treatment of the milk prior to inoculation, homogenization, incubation conditions, and handling of the ripened coagulum will all affect the body of the final milk product. Another major way to affect the body yogurt is through the addition of stabilizers such as gelatin, pectin or another polysaccharides. Stabilizers are added to the product to increase viscosity as well as to decrease susceptibility to syneresis (Schellhaass and Morris, 1985).

In dairy industry, the use of polysaccharide producing bacteria is of interest with respect to the improvement of body and texture of yogurt, in particularly in France and Netherlands where addition of plant or animal stabilizers is prohibited.

Curdlan is extracellular slime polysaccharide of Alcaligenes faecalis var. myxogenes strain 10C3 where found by Harada for the first time at 1965. Harada was isolated, purified this polysaccharide and he concluded that curdlan is shown to contain about 10% succinic acid, 70 - 80% glucose, and small amounts of galactose and mannose and it seems to have beta-glycosidic linkages (Takahashi et al., 1986). Curdlan is added to foods to improve their properties and is also used to make newfoods (Harada, 1992).

Although polysaccharide has been widely investigated, little information exists on how effect polysaccharide in reconstituted skim milk fermented by lactic acid bacteria. This study determined the influence of curdlan on the rheological properties and microstructure of acid milk curd by lactic acid bacteria fermentation.

Materials and Methods

Lactic acid bacteria

Lb. delbrueckii subsp. bulgaricus B-5b were obtained from Japan Milk Product Technology Association, Tokyo, Japan. During the course of the investigation the culture were routinely propagated in 10% RSM. The RSM was autoclaved at 121ºC for 15 min. and tempered to 37ºC prior to inoculation. A 0.1% inoculum was added to the RSM and the culture was allowed to incubate at 37ºC over night.

Preparation of acid milk curd

Acid milk curd was made from 10% RSM.　These milks were added with curdlan (Takeda Chemical Industries Ltd., Osaka, Japan) with different concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%), heated at 85ºC for 30 sec., cooled to 37ºC, inoculated with 1%(v/v) Lb. delbrueckii subsp. bulgaricus B-5b, and incubated at 37ºC for 16 h.

Rheometric properties

Curdlan added acid milk curds were studied toward the viscosity by using a viscometer (Tokimec Inc., Visconic ED-model). Steady shear rate of 1-100/s along with a MK 50 rotor assembly and NV sensor system operating at 25ºC, viscosity was expressed as millipascals per sec.

The hardness of acid milk curd was measured with a Sanwariken JK-T type rheometer (Sanwariken Ltd., Tokyo) as a curd knife cut the surface of milk curd. These measurement conditions were 1.44mm/sec in penetration speed of the curd knife, 18 cm/min in chart speed and 0.1V in sensitivity of a recorder.

Breaking energy of acid milk curd (stress x strain) was estimated on the basis of curd harness, cross section of the curd knife (0.1963 cm), length of milk curd (4.9363 cm), and penetration depth of the knife (0.4807 cm). Elastic modulus, stress/strain was a measure of elasticity in the instance of solid matter. The detailed calculation of breaking energy and elastic modulus for milk curd was described previously (Ohashi et al., 1983).

From the chart, value for X and Y were determined on the methods described by Ohashi et al., 1978), using the following equations and conditions.

Hardness = dyn

Breaking energy (dyn/cm²) = (G/A) x (a/L)

Elastic Modulus (dyn/cm²) = (G/A) / (a/L)

Where:

G = Hardness (dyn), A = Transversal area of knife,

a = Knife penetration, L = Height of sample.

Surface structure of Lb. delbrueckii subsp. bulgaricus B-5b

Appropriate dilutions of overnight Lb. bulgaricus subsp. Bulgaricus B-5b were spread on 5% skim milk agar plates with composition as follows:

Skim milk 50.0 g Yeast extracts 2.5 g

Peptone 5.0 g Glucose 1.0 g

Agar 15.0 g

These plates were incubated for 24-70 h at 37ºC. A 2-4 mm cube of the agar with a colony on it was cut from each plate. The sample were fixed in 2.5% glutaraldehyde solution for 24 h and post-fixed in 1% osmium tetraoxide solution for 5 h. The samples were then soaked in a series of ethanol distilled water solutions (50, 60, 70, 80, 90, 95, 99.5% (v/v) ethanol) as intermediate fluid. Samples were allowed to stay for 10 min in each concentration, dried in a Hitachi HCP-2 type critical point drier (Hitachi Ltd., Tokyo), coated with gold in a Hitachi E-1030 type sputtercoater ((Hitachi Ltd., Tokyo), and examined with a Hitachi S-4100 type scanning electron microscope (SEM, Hitachi Ltd., Tokyo) at an accelerating voltage of 1.0 kV.

microstructure of milk curd

A template was made by gluing 4x10 mm glass rods to the inside surface of a petridish cover. A 3% agar solution (60ºC) was pored 13 mm deep into the petridish. The template was then placed into the agar solution. The template was removed after the agar had solidified, which resulted in the formation of cylindrical pores in the agar. The coagulated milk curd was then pipeted into the pores. The surface was overlaid with 3% agar, which had been tempered to 45ºC. After the agar overlay had solidified, 6 mm cubes containing a single cylindrical pore of coagulated milk, were cut out of the agar. The agar cubes were fixed in 2.5% glutaraldehyde solution buffered at pH 7.0 with 0.1 M phosphate buffer, and then post-fixed in 1% osmium tetraoxide solution. Samples were dehydrated in a graded alcohol series as described above, and then dried in a Hitachi HCP-2 type critical point drying apparatus (Hitachi Ltd., Tokyo), coated with gold in a Hitachi JFC-1 type, ion type sputter coater (Hitachi Ltd., Tokyo), and viewed in a Hitachi S-4100 type scanning electron microscope (Hitachi Ltd., Tokyo) at an accelerating voltage 1.0 kV.

Results and Discussion

Rheological properties

Four kinds of rheological characterization have been examined in the experiment is viscosity, hardness, breaking energy and elastic modulus. However, there are many reason concerning with food quality control.

The effect of curdlan concentration on the apparent viscosity of milk curd was determined as shown in the fig. 1. Curdlan have two types of gels, 'low set gel' if its heated at 60ºC and 'high set gel' if its heated at 95ºC (Takahashi et al., 1986) or above 80ºC (Harada et al., 1991). We used heating at 85ºC for 15 sec. The viscosity of acid milk curd containing curdlan increased with increasing curdlan concentration. Viscosity of acid milk curd on addition of curdlan (0-1%) and heated at 85ºC for 15 sec followed by inoculation with 1% Lb. delbrueckii subsp. Bulgaricus B-5b and incubation at 37ºC for 16 h, increased from 64.7 to 342.2 mpa/sec. The viscosity increased slowly between 0% and 0.4% concentration of curdlan, and increased linearly with an elevation of concentration above 0.4% curdlan concentration. The increasing viscosity may be due to the fact that some casein, particularlyβ-casein, start to dissociate from the micelle, and dissolved casein molecules have a higher hydrodynamic volume. The addition of curdlan probably affected the hydrodynamic volume of casein micelle and acid milk curd formation. The use of curdlan in acid milk curd/yogurt increases the apparent viscosity and pH, however increasing pH is undesirable in yogurt making. In yogurt, lactic acid produced by the bacterial culture lowers the pH below the iso-electric point and induced coagulum of casein (Harwalkar and Kalab, 1986).

In the acid milk curd heated at 85ºC, hardness and breaking energy increased with increasing curdlan concentration (%). The breaking energy and hardness were not significant recognized between not-supplemented acid milk curd samples (0% of curdlan) and those with 0.2% curdlan concentration, and increased linearly with an elevation of concentration above 0.4% curdlan concentration. This result can be explained in the microstructure of acid milk curd. The size of clusters of casein micelle was increased with increasing curdlan concentration, and curdlan added-acid milk curd, the pore dimensions are diminished, and the density of the matrix was increased. The individual casein may self associate or form associations with other fractions through hydrophobic or electrostatic interactions.

Elastic modulus was demonstrated polynomial chart with increasing curdlan concentration. Compared with acid milk curd with 0% curdlan (no addition curdlan), in the acid milk with 0.2% curdlan, elastic modulus decreased until 0.87x10⁵ dyn/cm², from 2.58x10⁵ dyn/cm². Elastic modulus in acid milk curd with 0.4% of curdlan increased slowly from 0.76 to 2.50 x 10⁵ dyn/cm in the acid milk curd with 1% of curdlan.

Microstructure of acid milk curd

Scanning electron micrographs of the Lb. Delbrueckii subsp. Bulgaricus B-5b demonstrated that the surface appendages (slimy) is not present on the cell surface, indicated that the bacteria is non-ropy lactic acid bacteria.

Thickening agents are used to improve the texture, increase the firmness, and prevent syneresis in yogurt. This is important to help maintain good textural and visual properties during transportation and storage.

The application of SEM to explain rheological behavior when studying exopolysaccharide-producing bacteria was used to help understand the mechanism and influence the physical properties.

One of the reasons may be the relative difficulty of subjecting acid milk curd/yogurt to electron microscopy, particularly to scanning, because the fine yogurt network is very susceptible to electron beam damage (Kalab and Emmons, 1975). Fixation, washing, and subsequent drying preserved the spatial configuration of the protein in the initial network. Electron microscopy shows that the casein micelles were fused into chains and clusters, yet essentially retaining their globular shape in spite of the presence of bacteria possessing some proteolytic activity. The arrangement of the micelles and the formation of a protein skeleton created large free spaces inside the network, which are best seen under a scanning electron microscope. The casein micelles are easily distinguishable from lactic bacteria at magnifications over 2000x (Kalab and Emmons, 1975).

It has already been mentioned that in yogurt, casein particle chains are linked at random and firm a matrix with relatively uniform pores (Harwalkar and Kalab, 1986) filled with the liquid phase (whey). The milk curd containing curdlan, the pore dimensions are diminished, and the density of the matrix is increased. In heated induced milk gels with high concentrations of milk proteins (14-20%), the protein network consisted of casein micelles either connected by short bridges or fused into long chains a clusters (Kalab and Emmons, 1975).

Electron microscopy showed that yogurt consists of a protein matrix composed of chained and clustered casein particles. Chains are common in yogurt made from milk, which had been preheated to a minimum of 85ºC whereas large clusters of casein particles from the matrix of yogurt made from heated milk. Such a matrix is characterized by interstitial spaces (pores), the dimensions of which depend on the protein contention that matrix. The casein micelles in milk started to disintegrate as the pH of the milk reached 5.5 due to the production of lactic acid by the bacterial culture. The disintegration was most extensive at pH 4.8 but the proteins reaggregated into globular particles as the pH value was further decreased to 4.8 and lower (Harwalkar and Kalab, 1986). Yogurt made from heated skim milk, changes were not particularly conspicuous: individual micelles lost their sharp and smooth outlines and became ragged, grew somewhat in size and fused together into clusters and chains (Kalab and Emmons, 1975).

In a control acid milk curd containing no curdlan, only casein micelles and lactic acid bacteria (Lb. delbrueckii subsp. bulgaricus B-5b) composed the gel microstructure (Fig. 2A). In general, there was a slightly higher degree among linking casein micelles into chains and clusters in the presence of curdlan than in control acid milk curd (Fig. 2B-2F).

The lack of differences was attributed to the low curdlan concentration (0, 0.2, 0.4, 0.6, 0.8 and 1.0%) in the acid milk curd. Kalab and Emmons (1975) found that in yogurt contain gelatin may be visible to electron microscopy in yogurt containing 2% and 10% gelatin. Teggazt and Morris (1990) suggested that in ropy cultures, the EPS in attached to the bacterial cell surface and also interacts with the casein. When curdlan interacted with casein micelle they appeared as fluffy mass join by string between one-casein micelles and other casein micelles, and/or fluffy mass film in the surface of cluster of casein micelles. In acid milk curd with 0.6% curdlan concentration, string fluffy mass film were lower than acid milk curd with 1% curdlan concentration.

Conclusion

The result from this investigation can be concluded that curdlan increased rheological properties of acid milk curd. Scanning electron microscope of acid milk curd demonstrated that curdlan improve degree among linking casein micelles into chains and clusters in microstructure of acid milk curd.

Acknowledgments

We would like to thank Professor Tomio OHASHI and Professor Kiyoshi YAMAUCHI for supervision. We are grateful for the financial support Takeda Chemical Ind., Ltd., OSAKA for providing the samples of curdlan used in the experiments and Laboratory of Biochemistry and Technology of Miyazaki University, Japan.

References

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Figure 1. Relationships of curdlan concentration to the viscosity, harness, breaking energy and elastic modulus of acid milk curd, fermented by Lb. Delbruckii subsp. Bulgaricus B-5B at 37 ºC for 16 hours

A B

C D

E F

Figure 2. Microstructure of acid milk curd (10% skim milk). A) 0% of curdlan (control), B) added 2% of curdlan, C) added 0.4% of curdlan, D) added 0.6% of curdlan, E) added 0.8% of curdlan, F) added 1.0% of curdlan, a) Lb. Delbrueckii subsp. Bulgaricus B-5b, b) casein micelles