Glacier Journal Of Scientific Research issn: 2349-8498

Glacier Journal Of Scientific Research ISSN:2349-8498

Authors
1.	Dr.K.S.Gnanalaksshmi,Ph.D., Assistant Professor, College of Food and Dairy Technology, Koduvalli,Chennai-52. Ph.No:9445199034
2.	Dr.D.Baskaran,Ph.D., Professor and Head, Dept of Food Engineering, College of Food and Dairy Technology, Koduvalli,Chennai-52. Ph.No: 9176076199
3.	Dr.G.Sarath Chandra,Ph.D., Professor and Head, Pharmacovigilance Laboratory for animal; feed and Food safety. Madhavaram, Chennai-51. Ph:9444050644

Biosynthesis of Exopolysaccharide by lactic acid bacteria in low fat dahi.

Abstract

A study was conducted to identify Exopolysaccharides (EPS) producing lactic acid bacteria from the fermented milk products and its application as biothickener in dahi prepared from skim milk for the manufacture of highly viscous curd mass to impart desired qualities of fat rich dahi. A total number of 52 EPS ⁺ isolates were obtained from fermented milk products comprising of 27 mesophilic strains and 25 thermophilic strains. On the basis of morphological, bio-chemical and molecular tools, the above isolates were identified as Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus and Lactococcus lactis ssp. lactis. EPS production was found to be maximum for Streptococcus thermophilus followed by L. lactis ssp. lactis and Lactobacillus delbrueckii ssp. bulgaricus at the level of 1.726 ± 0.079 g/L, 1.517 ± 0.044 g/L and 1.08 ± 0.034 g/L in the respective order. The total protein content range observed between 21.312 mg/L and 25.979 mg/L. Dahi was prepared by using EPS⁺ isolate showed significantly lower syneresis than EPS^- NCDC 167 culture. Syneresis per cent of S.thermophilus inoculated dahi was lower when compared with other isolates.

Key words: Exopolysaccharides, LAB, low fat dahi

Introduction

Dahi is considered as the oldest Indian fermented milk product and is equivalent to Western yoghurt which contributes around 90% of total cultured milk products produced in India. . Lactic acid bacteria are widely employed in the manufacture of traditional fermented milk, in industrial fermentation processes and as starter cultures in the dairy industry. Apart from the production of lactic acid, , several strains of Lactic acid bacteria were able to secrete exopolysaccharides (EPS) .EPS are long-chain polysaccharides produced extracellularly mainly by bacteria and microalgae, EPS consists of branched, repeating units of sugars or sugar derivatives. In the food industry, EPS produced by LAB and other bacteria are used as food additives and act as viscosifiers, stabilizers, emulsifiers, or gelling agents to modify the rheological properties and texture of products. EPS produced by LAB have received much attention in recent years because of their contribution to the rheology and textural properties of food products. Health awareness among consumers generated more demands for fat-free Dahi in the Indian dairy market. However, milk fat contributed to the flavour, body and texture development of the dairy products, removal of fat leads to flavour and textural defects.

In this context, EPS producing LAB as ‘biothickeners’ can offer natural and more acceptable solution and can only be the preferred approach to many additives . These cultures meet the consumer requirement for products with low levels of chemical additive reduce the amount of total solids required without affecting the textural attributes and improves sensory properties.

Materials and Methods

Fresh skim milk obtained from the Dairy plant, Department of Dairy Science, Madras Veterinary College was used. Spray dried skim milk powder (Sagar brand) procured from local market was used as a medium for propagation of the lactic cultures which were used for preparing dahi. The following lactic culture was procured from National Collection of Dairy cultures, National Dairy Research Institute, Karnal, Haryana, India were used in the study. Mixed Dahi culture - NCDC 167 (Containing Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. diacetyl lactis and Leuconostoc citrovorum) was employed in the study.

A total number of 75, comprising of sixty dahi, five brands of yoghurt, five lassi and five shrikhand samples were collected aseptically from Street vendors, households, restaurants, hostels and organised sector in and around Chennai city. Appropriate dilution of dahi and other fermented milk products were made in sterile peptone water (0.1%) and plated out on MRS agar and incubated at 30ºC and 42º C for 24 to 48 hours. After incubation period colonies that showed mucoid appearance and /or formed long filaments when touched with the sterile tooth picks were randomly picked up and transferred into sterile skim milk tubes (10ml). The isolates were purified by streaking three times on Milk agar and MRS agar plates and well isolated colonies were selected. Typical mucoid colonies were enumerated and a total of 150 isolates were identified as LAB for further screening. A total number of 52 EPS⁺ isolates were obtained comprising of 27 mesophilic strains and 25 thermophilic strains. Dahi was found to be better source for isolation of EPS LAB as compared to other fermented milk products. The promising LAB isolates as selected based on titratable acidity, viscosity and sensory evaluation from genera i.e. Streptococcus, Lactococcus and Lactobacillus were subjected to further tests as per Bergys Manual to identify their species.Three isolates i.e. Streptococcus thermophilus, Lactococcus lactis ssp. lactis and Lactobacillus delbrueckii ssp. bulgaricus were identified as EPS producer.

Testing the ability of isolate for EPS production and Estimation of sugar in EPS.

150 isolates have been obtained for EPS extraction. 52 samples were analysed for EPS extraction by ethanol precipitation.

EPS extraction by ethanol precipitation by Savadogo et al. (2004)

Lactic acid culture

↓

Centrifuged at 11,000g for 10 mts.

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Cultures supernatant collection.

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Addition of two volumes of cold ethanol and stored overnight at 4° C

↓

Centrifuged at 2500 g for 20 mts.

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Precipitated material was resuspended in demineralized water

and mixed with 2 volumes of cold ethanol.

↓

Centrifuged 2500 g and pellets dried at 100º C

(Total carbohydrate was estimated by using spectrophotometer by Anthrone method)

The sugar content in EPS was estimated by Anthrone method described by Southgate (1991). Fresh anthrone reagent was prepared every time by weighing 0.2 g anthrone and adding to 100 ml of 95% ice cold concentrated sulphuric acid. Low fat dahi was prepared as per the procedure out lined by Aneja et al., (2002). All three EPS Positive lactic isolates and Non EPS producing (mixed Dahi culture NCDC 167 obtained from National collection of Dairy cultures, Karnal, India) cultures were used to ferment milk.

Results and Discussion

Isolation of EPS producing Lactobacillus sp and EPS production.

Dahi and other fermented milk products viz., yoghurt, lassi, shrikh and were plated on milk agar and incubated at 30 and 42°C for 72 hrs. Recovery of colonies of lactobacilli was found to be appreciable in MRS agar and milk agar plates. Streak plate method was observed as a suitable method for isolation of lactobacilli from fermented milk products. A total of 150 mucoid ropy colonies were transferred into sterile skim milk. The skim milk which showed clear fermentations was selected and under fermentations was discarded. The cultures were purified by repeated streaking to obtain pure cultures. A total number of 52 EPS⁺ isolates could be obtained comprising of 27 mesophilic and 25 thermophilic strains. Dahi was found to be better source for isolation of EPS LAB as compared to other fermented milk products. EPS production was found to be maximum for Streptococcus thermophilus followed by L. lactis ssp. lactis and Lactobacillus delbrueckii ssp. bulgaricus at the level of 1.726 ± 0.079 g/L, 1.517 ± 0.044 g/L and 1.08 ± 0.034 g/L in the respective order.

Whey separation in Non EPS and EPS producing culture inoculated dahi

The data with respect to whey separation in dahi samples inoculated with EPS⁺ and EPS^- cultures are given in Table 1.

Streptococcus thermophilus (T₁), Lactobacillus delbrueckii ssp. bulgaricus (T₂), and Lactococcus lactis ssp. lactis (T₃) exhibited syneresis per cent of 20.70, 21.48 and 20.89 as compared to 23.94 per cent in EPS^- culture maintained as control.

There was a significant (P < 0.05) difference noticed in whey separation between control EPS^- culture inoculated dahi sample (T₀) and EPS⁺ culture inoculated dahi samples (T₁,T₂ and T₃).

There was a significant reduction (P < 0.05) in whey separation observed when EPS⁺ Streptococcus thermophilus (T₁), Lactococcus lactis ssp. lactis (T₃) and Lactobacillus bulagaricus (T₂) were used for Dahi preparation in the descending order as compared to control.

With decreasing fat concentration in dahi samples the wheying off significantly increased. Higher fat concentration improves the gel formation of dahi which may result in firm, well set and higher viscous curd as reported by Yadav et al. (2005).

The susceptibility to whey separation was reduced in lowfatdahi made by EPS⁺ cultures due to the ability of EPS to bind significant amount of water. Similar findings were observed in most of the previous studies conducted by Marshall and Rawson (1999) and Doleyres et al. (2005).

EPS⁺ cultures were capable of producing gel with significant ropiness in fermented milk products upon inoculation which restricts the release of free water through formation of casein network. This observation was noticed by Guzel-Seydim et al. (2005) who reported that whey separation in yogurt decreased significantly with ropy cultures when fermented at 35°C than with non-ropy cultures at the same incubation temperature.

Skim milk with 0.37 per cent fat concentration was used for all the inoculations and treatments due to which the wheying off had been expected to increase significantly. In contrary, due to utilization of EPS⁺ cultures, the syneresis was not only kept under check but also reduced significantly for Streptococcus thermophilus, Lactococcus lactis ssp. lactis followed by Lactobacillus delbrueckii ssp. bulgaricus, in that order. Higher fat concentration improves the gel formation of dahi which may result in firm well set and higher viscous curd.

EPS thus have an important role as natural biothickening agents to improve the rheology of a fermented product and as physical stabilizers to bind water and limit whey separation as reported by Ruas-Madiedo et al. (2002a) who stated that the addition of EPSs as texturizers and stabilizers increases the viscosity of the product, and they interact with proteins and micelles by binding water, hence supporting the rigidity of the casein network. EPSs could decrease syneresis and improve product stability.

Sensory scores of Non EPS and EPS producing culture inoculated lassi

The data with respect to appearance, flavour, body and texture, sourness and overall acceptability for of EPS^- and EPS⁺ culture inoculated dahi samples stored at 5°C (Table 9) revealed that the panel of six trained judjes were given fresh dahi samples for evaluation. The scores given by the judges for individual attributes were computed. There was a significant difference noticed between control and EPS⁺ culture inoculated dahi samples for all the attributes. This coincides with the findings of Behare et al. (2009).

The sensory scores obtained for different experimental samples of dahi which was prepared with EPS⁺ culture were attractive and had uniform body with smooth, yellowish, glossy appearance without any wheying off on surface of on the product. The body and texture of T₁, T₂, T₃ dahi was smooth and glossy while the cut surface was firm and free from crack and gas bubbles.

Sensory attributes were improved by use of EPS⁺ cultures which enhanced mouth thickness, shininess and tended to be creamier than the dahi made by non EPS^- cultures.

Conclusion

In conclusion, microbial exopolysaccharides derived from LAB attracts attention and importance due to their GRAS status and hence an attempt was made to isolate EPS positive isolates of LAB from fermented milk products which was resulted in the identification and characterization of three promising EPS⁺ cultures viz. S.thermophilus, Lactobacillus delbrueckii ssp. bulgaricus and Lactococcus lactis ssp. lactis. EPS⁺ cultures have shown significant improvement in rheological and sensory properties and played a vital role to control syneresis in fermented milk products as compared to EPS^- cultures.

References:

Behare, P., Singh,R and Singh, R.P.(2009). Exopolysaccharide - producing mesophilic lactic cultures for preparation of fat free Dahi-an Indian fermented milk. J. Dairy Res., 76: 90-97.

Dolyeres, Y., Schaub,L and Lacroix,C (2005). Comparison of the functionality of exopolysaccharides produced insitu or added as bioingredientson yoghurt properties.J.Dairy Sci., 88: 4146-4156.

Guzel-Seydim, Z. B., Sezgin, E. and Seydim, A. C. (2005). Influences of exopolysaccharide producing cultures on the quality of plain set type yogurt. Food Control. 16: 205-209.

Marshall, V. M. and Rawson, H. L. (1999). Effects of exo polysaccharides-producing strains on thermophilic lactic acid bacteria on the texture of stirred yoghurt. Int. J. Food Sci. Technol., 34:137-143.

Ruas-Madiedo, P., Hugenholtz,j, and Zoon,P(2002a). An overview of the functionality of exopolysaccharides produced by lactic acid bacteria . Int. Dairy J., 12: 163-171.

Savadogo, A ., Outtara, C.A.T, Savadogo, P.W, Barro, N, Outtara , A. S.and Traore, A.S(2004). Identification of exopolysaccharides-producing lactic acid bacteria from Burkino Faso fermented samples. African J. Biotechnol., 3 : 189-194.

Yadav, H., Jain,S and.Sinha, P.R (2005). Preparation of low fat probiotic dahi J. Dairying Food Home Sci., 24:172-177.

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