Introduction

Yoghurt is a coagulated milk product which is produced due to the fermentation activity of Lactobacillus bulgaricus and Streptococcus thermophilus like lactic acid bacteria in the milk and milk products [1]. Lactic acid bacterial collection which use in the yoghurt production is called yoghurt starter culture and most of the industrial yoghurt starter cultures are defined mixed cultures [2]. These defined cultures can be DVS (Direct vat set cultures) and bulk cultures [3]. Defined yoghurt starter cultures consist with various kinds of microorganisms as Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Streptococcus thermophilus Lactobacillus lactis and Bifidobacterium sp. Proto cooperation among lactic acid bacteria gives proper chemical and physiological characters to the yoghurt [4]. Proto cooperation is stimulation of each other’s growth by exchanging metabolites produced by each of these lactic acid bacteria [5]. In fact, S. thermophilus utilizes amino acids in milk and produce folic acid. This folic acid stimulates the growth of L. bulgaricus. Metabolic activity of each of these bacteria enhances their growth rates due to the available compound in the medium and produced compounds to the medium. As a result of these metabolic potentials, growth curve of S. thermophilus consists with two exponential phases separated from transition phase (Figure 1). Due to the created favorable environment by S. thermophilus, L. bulgaricus starts their exponential growth [6]. For well proto cooperation, microbial ratio of the lactic acid bacteria in the yoghurt starter culture is very important. As well as, proper proto cooperation controls the post acidification when yoghurt store under the cooling temperature for long time. Survival ability of lactic acid bacteria in cooling temperature of the yoghurt creates excess acidity in the yoghurt and it is called post acidification [7]. Due to the high acidity development in the yoghurt, it causes some leakages of whey proteins which are called syneresis [8]. These qualitative defects in the yoghurt structure reduce the shelf life of the yoghurt sample. Post acidification depends on the type of strain, microbial ratio in the yoghurt starter culture, the storage temperature, and the storage time. Post acidification manipulation can be done changing the microbial ratio and it is increased the shelf life of the yoghurt. Sri Lankan yoghurt producers basically depend on the bulk yoghurt starter cultures. The microbial ratio of the bulk yoghurt starter culture determines the quality and shelf life of the yoghurt. The better proto cooperation among the lactic acid bacteria build up the proper chemical and physical qualities in the yoghurt and control the postacidification effect of the yoghurt. Therefore, this attempt is to select the best lactic acid microbial ratio which can produce yoghurt with proper chemical and physical properties and low post acidification to enhance the shelf life of yoghurt.

Method and Materials

Determination of LB: ST ratio of currently using bulk culture

L. bulgaricus to S. thermophilus ratio of the currently using bulk culture of Milco (pvt) Ltd in Sri Lanka was determined following serial dilution and pour plate method using modified Elliker medium.

Isolation of L. bulgaricus and S. thermophilus

L. bulgaricus and S. thermophilus bacterial species were isolated from the currently using bulk culture in the milk factory. Moreover, L. bulgaricus in the bulk yoghurt starter culture was isolated into modified MRS medium and S. thermophilus was isolated into modified M17 medium. Then pure cultures were prepared. LB1: ST1 ratio, LB1.5: ST1.5 ratio and LB1:ST1.5 yoghurt starter cultures production L. bulgaricus and S. thermophilus were separately inoculated into autoclaved skim milk samples and incubated at 41o C for 24 hrs to take high dense cultures for LB1:ST1 ratio yoghurt culture preparation. After both samples reached to 0.5 OD level, 50 ml of each sample was added into 4.95 L of autoclaved skim milk sample and it was incubated at 41o C for 24 hrs and after the incubation period colony count was taken and it was kept under cooling conditions for 10 hrs period. Same method was followed up to prepare LB1.5:ST1 ratio culture only by changing the mixing volumes as 60 ml from Lactobacillus bulgaricus and 40 ml from Streptococcus thermophilus. The sample was kept at 41o C for 24 hrs and then it was kept in cooling room for 10 hrs time period. Same method was followed to produce

LB1: ST1 ratio, LB1.5: ST1.5 ratio and LB1:ST1.5 yoghurt starter cultures production

L. bulgaricus and S. thermophilus were separately inoculated into autoclaved skim milk samples and incubated at 41o C for 24 hrs to take high dense cultures for LB1:ST1 ratio yoghurt culture preparation. After both samples reached to 0.5 OD level, 50 ml of each sample was added into 4.95 L of autoclaved skim milk sample and it was incubated at 41o C for 24 hrs and after the incubation period colony count was taken and it was kept under cooling conditions for 10 hrs period. Same method was followed up to prepare LB1.5:ST1 ratio culture only by changing the mixing volumes as 60 ml from Lactobacillus bulgaricus and 40 ml from Streptococcus thermophilus. The sample was kept at 41o C for 24 hrs and then it was kept in cooling room for 10 hrs time period. Same method was followed to produce LB1:ST1.5 ratio culture only by changing the mixing volumes as 40 ml from Lactobacillus bulgaricus and 60 ml from Streptococcus thermophilus. The sample was kept at 41o C for 24 hrs and then it was kept in cooling room for 10 hrs time period. Final microbial ratio of each new yoghurt starter culture after the incubation period was determined following the serial dilution method.