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

7:06 AM |


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 85oC selama 30 detik, diinokulasi dengan 1% Lb. delbrueckii subsp. bulgaricus B-5b dan diinkubasi pada 37oC 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

Harada T., Kanzawa Y., Kanenaga K., Koreeda A. and A. Harada.  1991. Electron microscopic studies on the ultrastructure of curdlan and other polysaccharides in gels used in foods. Food structure, 10: 1-18.

Harada T. 1992.  The story of research into curdlan and the bacteria producing it. Trends in glycoscience and glycotechnology, 4(17): 309-421.

Harwalkar V.R. and M. Kalab. 1986. Relationship between microstructure and susceptibility to syneresis in yogurt made from reconstituted nonfat dry milk. Food microstructure, 5: 287-294.

Kalab M. and D.B. Emmons. 1975. Milk gel structure. V. Microstructure of yogurt in relation to the presence of thickening agents. J. of dairy research, 42: 453- 458.

Ohashi T., Haga S., Fujino H., Taniyama S., Yamaguchi K. and T. Akiyama. 1978.  Studies on the physical properties of milk and milk product: on the hardness, breaking energy and elastic modulus of milk rennet curd. Nippon shokuhi kogyo gakkaishi, 25 (10): 38-40.

Ohashi T., Nagai S., Masaoka K., Haga S., Yamaguchi K. and N.F. Olson. 1983.  Physical properties and microstructure of cream Cheese. Nippon Shokuhin kogyo gakkaishi, 30(5): 303-307.

Schellhaass S.M. and H.A. Morris. 1985.  Rheological and scanning electron examination of skim milk gels obtained by fermenting with ropy and non-ropy strains of lactic acid bacteria. Food microstructure, 4: 279-287.

Takahashi F., Harada T., Koreeda A. and A. Harada. 1986.  Structure of curdlan that is resistant to (1→3) ß-D-glucanase. Carbohydrate polymers, 6: 407-421.

Teggazt J.A. and H.A. Morris. 1990.  Changes in the rheology and microstructure of ropy yogurt during shearing. Food structure, 9: 133- 138.








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











Figure 3.  Microstructure of acid milk curd (10% reconstituted skim milk) with addition of 0.6% curdlan (18,000x).














Figure 4.  Microstructure of acid milk curd (10% reconstituted skim milk) with addition of 1.0% curdlan (18,000x).

1 Staf Jurusan Produksi Ternak, Fakultas Peternakan Universitas Hasanuddin

Di publikasi pada BIPP (terakreditasi) VIII (2): 142 - 147 (2001)

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Posted by Ratmawati Malaka's Blog

Exopolysaccharide production by Lactbacilus bulgaricus

6:51 AM |



Exopolysaccharides production by Lactobacillus bulgaricus:
 A review
Ratmawati Malaka[1]),  Effendi Abustam1), Metusalach[2]), and Amran Laga[3])

ABSTRACT
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            The article explain exopolysaccharides production by Lactobacillus bulgaricus by literature studied. The production of exopolysaccharides (EPS) by Lactobacillus bulgaricus, chemical composition, structure, synthesis and application are outlined.  Production of EPS by bacteria is dependent on the temperature and pH of the medium in terms of carbon, nitrogen, mineral and vitamin content.  Optimal EPS production by L. bulgaricus were at 30oC.  Most of researches concluded that L. bulgaricus produced EPS between 130 – 330 mg/l in suitable environment and medium.  Composition of EPS mainly composed glucose, galactose and rhamnose in variative ratio, but galactose often dominant comparison in another monosaccharide.   Application of EPS in food can increase the quality of product. On the other hand, the possibility that this EPS may be involved in antitumor activity and another pharmaceutical agent need more confirmation.

Keyword:  Lactobacillus bulgaricus, Exopolysaccharide, Antitumor activity, Pharmaceutical agent,  Food addictive, Lactic Acid Bacteria
 
INTRODUCTION
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            Exopolysaccharides are produced by great variety of bacteria including lactic acid bacteria (LAB) (Broatbent et al., 2003).  The name exopolysaccharides (EPS) as proposed by Sutherland (1972) provides a general term for all these forms of bacterial polysaccharides found outside the cell wall and will be used in this article. 
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            Microorganism produce EPS in two distinct forms: ropy EPS or loose slime that is excreted into surroundings, and capsular EPS that remain adhered to cell surface creating a discrete covering (Zisu and Shah, 2003).  The role of EPS to cell bacteria is not clearly defined.  In the last decade, an increasing interest was observed in the EPS produced by the good-grade LAB, such as L. bulgaricus as starter culture of fermented milk. These polysaccharides play an important role in the rheology, texture and mouth feel of fermented milk. The objective of this article is to review the EPS production by L. bulgaricus.
PRODUCTION
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The productions of EPS are found in many species of both Gram-positive and Gram-negative bacteria and have been the subject of numerous investigations in varied fields of interest (Schellhass, 1983).  Soil scientists have an interest in EPS doe to the importance of these polysaccharides to soil properties and the possible role of EPS in the symbiotic relationship between different species of Rhizobium and legumes.  The industrial microbiologist has become interested in exploiting microbial EPS production due to increasing interest and need for novel polysaccharides (Malaka, 1997).  Utilizing microorganisms to produce EPS may offer both economic and availability advantage over traditional plant and marine sources. 
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            Most mucoid bacteria produce EPS under all cultural conditions, but the growth conditions must be optimized for maximal production. Lactobacillus bulgaricus capable of forming 250 mg/l of EPS on milk ultra filtrated, while as much as 350 mg/l had been found in milk  (Cerning, 1990; Grobben et al., 1995). 
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            Production of EPS is dependent on the temperature and pH of the medium as well as composition of the medium in terms of carbon and nitrogen source, and mineral and vitamin contents (Gamar-Nourani et al., 1998; Grobben et al., 2000).  The effect of temperature and pH on EPS production is highly variable and is dependent on the strain used and the experimental conditions.  Some workers have found EPS production to be optimal at low temperature (Marshall et al., 1995; van den Berg et al., 1995), while others have shown EPS production to be favored at much higher temperatures (Grobben et al., 1995; De Vuyst et al., 1998).  The optimum pH for EPS production generally ranges between 5 and 7 (De Vuyst et al., 1998).  Schellhaass (1983) reported that growth at pH 6.2 and 6.0 resulted in more polysaccharide production with L. bulgaricus RR  in skim milk dialysate than growth at pH 5.8 and 5.5. Maintenance of higher pH might result in increased exopolysaccharide production by increasing the time the culture was in log phase.  Higher pH results in a longer stationary phase, which would decrease peptidoglycan and teichoic acid synthesis and could result in increased exopolysaccharide production. Malaka and Abustam (2004) have shown that optimal EPS production for L. bulgaricus were at 30oC.  Shellhaass and Morris (1985), Malaka and Abustam (2004) indicated that lower incubation temperature resulted in slower growth rate, which promoted longer logarithmic and stationary phases.  Gassem et al. (1997) reported that when lactose was hydrolyzed during fermentation, glucose was utilized by the bacteria and galactose remained in the medium. The composition of the medium (Marshall et al., 1995) and carbon source (Gamar et al., 1997) are shown to increase EPS production.  Grobben et al. (1998) have been shown that EPS production by L. bulgaricus enhanced by multiple-vitamin omission. 
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            Many studies showed a decrease in the total EPS amount when incubation times were increased (Cerning et al., 1992; De Vuyst et al., 1998; Ganzel and Novel, 1994; Mozzi et al., 1996).  The decreased EPS level upon prolonged fermentation may be due to an enzymatic degradation (Cerning, 1990; De Vuyst et al., 1998) or a change in the physical parameters of culture (De Vuyst et al., 1998).  Ganzel and Novel (1994) suggested some reversible DNA rearrangements leading to different cell types which differ in exopolymer production capabilities.  However, the possible relationship between EPS production and the factors contributing to EPS degradation have not been investigated yet.
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            Bouzar et al. (1995) have shown that pink colonies of L. bulgaricus produced the highest amount (130 mg/l), the white colonies produced the lowest (70 mg/l), and the parental strain produced an intermediate amount (110 mg/l) of EPS.  Approximately half of the EPS was produced during the exponential growth phase; while Malaka and Abustam (2004) have shown that EPS was produced during the last exponential growth phase go in the stationary growth phase.  Lactobacillus delbrueckii ssp. bulgaricus RR can be grown in protein-hydrolyzed whey and produce EPS (between 313-330 mg/l), although the bacteria is typically weakly proteolitic and grows poorly in un supplemented media (Briczinski and Roberts, 2002).  The level of EPS production obtained by Toba et al. (1992) through fermentation of skim milk by L. delbrueckii sups. bulgaricus NCFB 2483 were 150 mg/l, by Cerning et al. (1986) through fermentation of skim milk with 1% casein by CNRZ 737 were 424 mg/l, and by Kimmel et al. (1998) through optimizing in semi-defined medium by strain RR were 354 mg/l.  Mozzi et al. (1995) have been shown that L. bulgaricus was the best EPS producer among the bacteria assayed (Streptococcus thermophilus, Lactobacillus acidophilus), an amount of 121 mg/l being obtained at 37oC after 24 h of fermentation.
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            Petry et al. (2000) developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and EPS production of two strains of L. delbrueckii subsp. bulgaricus.  The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as urotic acid and the carbon source.  EPS production was greatest during the stationary phase.  For strain CNRZ 416 without pH-controlled conditions, 10 g of glucose per liter seemed to be optimal carbon source concentration for the highest EPS yield.  The EPS yield of strain CNRZ 1187 appeared not to be affected by the sugar concentration of the medium.  The EPS yield of the CNRZ strain was 175 mg/l.

COMPOSITION AND STRUCTURE
           
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            The findings of numerous researches agree in as much as EPS indicate galactose and glucose to be released after hydrolysis of the polymer (Schellhaass, 1983; Cerning et al., 1986), but the molar ratios reported are very different.  Already by Cerning et al. (1986) investigated the composition of slime secreted by a strain of L. bulgaricus and reported that galactose was the most frequently encountered monomer within the polysaccharide, glucose and rhamnose are present in smaller amounts.  Gas liquid chromatography confirmed the HPTLC identification and revealed that the polymer is composed of galactose, glucose and rhamnose in a molar ratio of approximately 4: 1:1.  The polymers produced by the ropy and non-ropy strains have the same sugar components.  The elution volume of the EPS from the CL Sepharose $B column was almost the same as that of dextran of molecular weight of 488.000 Da and a single sharp peak was observed indicating that the EPS consisted of a single polymer.
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There exist three important groups of EPS produced by LAB: (i)α-glucans, mainly composed of α-1,6- and α-1,3-linked glucose residues, namely, dextran produced by Leuconostoc mesenteroides subsp. Mesenteroides and L. mesenteroides subsp. Dextranicum and mutants produced by Streptococcus mutants and S. sobrinus; (ii) fructans, mainly composes of β-2,6-linked fructose molecules, such as levan produced by S. salivarius; (iii) heteropolysaccharides produced by mesophilic (Lactococcus lactis subsp. Lactis and L. lactis subsp cremoris) and thermophilic (L. delbrueckii subsp. bulgaricus, L. helveticus, and S. thermophilus) LAB (Pham et al., 2000).
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            The sugar components of EPS L. bulgaricus are most frequently galactose, glucose and very often rhamnose (Cerning, 1990); galactose and small quantities glucose and rhamnose (Grobben et al., 1997).  The EPS from strain L. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain CNRZ 416 contained galactose, glucose and rhamnose (Petry et al., 2000).
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            These heteropolysaccharides are composed of linear and branched repeating units varying in size from tetra – to heptasaccharides.  The final EPS of high molecular mass   (1 x 106 – 2 x 106 Da) is formed by polymerization of several hundred to a few thousands of these repeating units (Petry et al., 2000).  L. bulgaricus yielded EPS of molecular weight of approximately 500.000, that contained galactose, glucose, and rhamnose in a molar ratio of approximately 4:4:1 (Cerning et al., 1991); 5 : 1 : 1 with predominantly 1, 4 and 1, 3 linkage patterns (Gruter et al., 1993).

BIOSYNTHESIS

            Zourari et al. (1992) suggested that L. delbrueckii subsp. bulgaricus, lactose uptake takes place via a lactose permease.  Inside the cell, β-galactosidase cleaves lactose to form glucose and galactose.  The latter is exchanged with lactose via a lactose-galactose antiport system.  Malaka and Abustam (2004) reported that during the early exponential phase of growth of L. delbrueckii subsp. bulgaricus RR, there was no EPS biosynthesis.  Production of EPS started at the end of exponential phase, but most EPS was produced at the end of growth and approximately 90% of glucose was consumed in the stationary phase (Petry et al., 2000).
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            Exocellular polysaccharides are synthesized in different growth phases and under a variety of conditions, depending on the organism studied (Cerning, 1990).  The process of synthesis involved can be divided into two principal categories based essentially on the site of synthesis and the nature of the precursors, i.e. synthesis outside the cell or at the cell membrane (Sutherland, 1972). 
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In many cases, research interest in biosynthesis of bacterial heteropolysaccharides has been driven by the knowledge that these polymers played an important role in virulence.  Thus, EPS biosynthesis is relatively well defined in Gram-negative pathogenic bacteria such as Escherichia coli, Salmonella, and Haemophilus influenza.  In these gram-negative systems, assembly of the basic repeating unit occurs at the cytoplasmic membrane and involves sequential transfer of sugar nucleotide diphospho precursors to an isoprenoid lipid carrier, undecaprenyl phosphate (Sutherland, 1972).  Once the basic repeating unit is assembled, the lipid-linked intermediates are usually translocated across the membrane and polymerized outside of the cell.  Then, the EPS may be covalently linked to the cell surface to form a capsule, or released into the medium as slime (Broadbent et al., 2003).
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Functional genetic studies and homology comparisons of the EPS-related gene products from some species lactic acid bacteria (maybe included L. bulgaricus) strongly suggest that cellular mechanisms for EPS synthesis in these species are similar to Gram-negative systems that require formation of lipid-linked intermediates (Stingele et al., 1996; Broadbent et al., 2001).  Work by Stingele et al. (1999) has shown that EPS biosynthesis in S. thermophilus involved a lipid carrier, upon which the sugar monomers that comprise the basic repeating unit are assembled.  The biosynthesis of EPS by L. bulgaricus has not been reported yet, but maybe similar with S. thermophilus.  The first step is catalyzed by Eps, a galactosyl-1 phosphate transferase that attaches galactosyl-phosphate to a lipid-phosphate carrier.  This reaction is followed by the action of three other glycosyltransferases, termed EpsG, EpsI, and EpsF, which sequentially attach N-acetylgalactosamine, glucose, and a branching galactose moiety to the basic repeating unit.  Unfortunately, very little is known about EPS polymerization, translocation, or slime formation by L. bulgaricus or any other Gram-positive bacteria.  Amino acid homologies between enzymes involved in these processes in Gram-negative and Gram-positive bacteria suggest that they occur via similar mechanism, but this hypothesis awaits experimental confirmation (Broatbent et al., 2003).
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            Cellular EPS biosynthesis is, therefore, an energy-intensive process that involves enzymes for production of sugar nucleotide precursors, a glycosyl1-1-phosphate transferase that transfers the first sugar group onto a phosphorylated carrier lipid, one or more glycosyltransferases that sequentially add new sugar groups to the growing repeat unit, and additional enzymes that participate in EPS gene regulation, membrane translocation, and polymerization/ chain length determination function (Cerning, 1990).  These reactions commonly involve additional proteins with function that are not unique to this process (Sutherland, 1972).
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            As noted previously, the lipid carrier for EPS assembly in Gram-negative bacteria is undercaprenyl phosphate, and participation of this molecule in EPS synthesis by LAB may be significant because it is also required for assembly of peptidoglycan, teichoid acids, and lipoteichoid acids (Cerning, 1990).  Specifically, if the cellular concentration of UDP is limited, then competition for lipid carrier may be a key limiting factor in EPS biosynthesis.  This hypothesis is supported by the frequent observation that incubation under conditions that stimulate bacterial growth and division reduce EPS production, while growth conditions which reduce for new cell wall synthesis (such as incubation at a suboptimal temperature) tend to enhance EPS production (Sutherland, 1972; Cerning, 1995; Malaka and Abustam, 2004).
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            Competition for sugar nucleotide precursors is another potential limitation to high EPS yields from EPS-producing LAB (Boels et al., 2001).  Many of the sugar nucleotide precursors used for EPS occur as intermediates in sugar catabolic pathways or, like UDP, are needed for the assembly of peptidoglycan and other glycan-containing cellular polymers (Broadbent et al., 2003).  Because low levels of sugar precursors are limiting factors on EPS yields (unpublished data), greater understanding of L. bulgaricus metabolic flux in sugar metabolism may provide new strategies to enhance EPS production.  Several researchers have demonstrated a correlation between EPS production and intracellular activities on enzymes from Leloir (Galactose-metabolism) and glycolytic pathways, but strain to strain variations are also apparent (Escalante et al., 1998; Levander and Radstrom, 2001).  Although the basis for these differences is not yet clear, recently in our research (unpublished data) showed such knowledge can be used to enhance EPS production in L. bulgaricus by metabolic engineering of central carbon metabolism.

APPLICATION OF EPS

            EPS have a variety of industrial applications, including their use as biothickeners in foods.  EPS produced by LAB contribute significantly to the structure and viscosity of fermented milk product (Malaka, 1997; Malaka et al., 1996; Malaka et al., 1997; Malaka and Baco, 2000; Boels et al., 2003). Polysaccharides may function in foods as viscosifying agents, stabilizers, emulsifiers, gelling agents, or water binding agents (van den Berg et al., 1995).  EPS of bacteria origin have unique rheological properties because of their capability of forming very viscous solutions at low concentrations and their pseudoplastic nature (Yang et al., 1999).  The EPS produced by food-grade LAB have been considered as a new generation of food thickeners to improve the rheological properties of foods (Robijn, 1996).  Perry et al. (1997) reported that using EPS starter cultures consisting of specific strains of L. bulgaricus increases moisture retention and improve the melt of lower-fat mozzarella cheese.  Hess et al. (1997) demonstrated that yoghurt made with EPS-producing  strains of L. delbrueckii subsp. bulgaricus were less susceptible to syneresis, required less force to penetrate the gel, and were more extensible than yoghurt made with nonropy strain (non EPS-producing strains).  The EPS appear to interact with the surface of bacterial cells and is associated with the protein matrix, affecting the viscosity and stability of the milk gel (Schellhaass and Morris, 1985).
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 Furthermore, several reports indicate that they can confer health benefits on consumers arising from their immunogenic and cholesterol-lowering properties (Hess et al., 1997).  As well as live bacteria (probiotics), EPS can improve intestinal balance to promote health; dietary carbohydrates may function as prebiotics, beneficially affecting the colonic microflora (Yang et al., 1999).
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Oda et al. (1983) reported an antitumor EPS produced by L. helveticus.  The slime produced by Bifidobacterium adolescentis had immunomodifying effects on mouse splennocytes (Gomez et al., 1988).  Kitazawa et al. (1992) showed that the slime-forming Lc. Lactis subsp. cremoris KVS 20 had antitumor activity, and the slime contained strong B-cell dependent mitogenic substances.


CONCLUSIONS

5
 
            Production of EPS by L. bulgaricus is growth-associated, and it is influenced by medium compositioc, culture temperature and pH.  Variations in carbon sources in growth medium resulted in different yields of EPS produced by L. bulgaricus, product range from 110 mg/l until 330 mg/l.
10
 
            The EPS L. bulgaricus was shown to consist of linear and branched repeating units varying in size from tetra – to heptasaccharides.  The sugar component of the EPS is most frequently galactose, glucose and very often rhamnose in a molar ratio of approximately 4:4:1 (Cerning et al., 1991); 5:1:1 with predominantly 1,4 and 1,3 linkage patterns (Gruter et al., 1993). 
            The EPS produced by foodgrade LAB, such as L. bulgaricus have been considered as a new generation of food thickeners to improve the rheological properties of foods.  On the other hand, the EPS have potentials pharmaceutical prospect as antitumor activity, or antivirus would be develop more extensive in the future time.

ACKNOWLEDGEMENT
I am indebted to  Prof. Sudirman Baco for his suffort and reading the manuscript.
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[1]) Laboratory of Animal Product Technology, Faculty of Animal Husbandry, Hasanuddin University, Indonesia, Telp : 0411-583111, fax 0411-586492.
[2]) Laboratory of Fish Product Technology, Faculty of Fishery, Hasanuddin University, Indonesia
[3]) Laboratory of Agriculture Technology, Faculty of Agriculture, Hasanuddin University, Indonesia

Dimuat pada Jurnal Instek, 2 (2): 152 - 164 (2005)

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