Isolation and characterization of exopolysaccharide‐producing strains of Lactobacillus bulgaricus from curd

Abstract Curd is the most widespread traditional fermented milk product used by a large population and is a good source of vitamin B, protein, and calcium. In this study, the isolation of exopolysaccharide (EPS)‐producing strains of Lactobacillus delbrueckii subsp. bulgaricus from curd samples was carried out. Identification of EPS‐producing strains was done by Gram staining, catalase activity, sugar fermentation test, API 50 CHL, and PCR analysis. These EPS‐producing strains were subjected for the estimation of technological properties such as titratable acidity, curdling time, acidification rate, and texture. The strains best in their technological properties were selected for the production of yogurt in combination with EPS‐ or non‐EPS‐producing strains of Streptococcus thermophilus. The EPS concentration range was from 41 to 268 mg/L in the yogurt. The highest value of EPS concentration was detected in S. thermophilus and non‐EPS‐producing Lb. bulgaricus after 14 days of storage.


| Exopolysaccharide-producing strain selection
Eight strains of Lb. delbrueckii ssp. bulgaricus were screened after biochemical and molecular tests from indigenous curd. These isolated strains were subjected for their EPS production by inoculating them into sterilized (121°C for 15 min) 10% skim milk and incubated at 42°C for 24 hr. Then, 2% of fermented sample was added in freshly prepared MRS broth and incubated at 42°C for 24 hr for EPS estimation.

| Isolation
Isolation of EPS was done by the method described by Rimada and Abraham (2003). Ten milliliters of sample was taken from the fermented bottle and heated in boiling water bath at 100°C for 15 min to dissolve the polysaccharides that are attached to cells and to inactivate the enzymes. After cooling, the samples were centrifuged at 15941 g at 20°C for 10 min to remove cells and 17 ml of 85% trichloroacetic acid was added for 100 ml of sample and cooled at 4°C, and then centrifuged at 8,000 rpm for 10 min to remove protein contents from samples. Precipitation of EPS from samples was provided using cold ethanol (−20°C, 1:3). Samples were stored at 4°C for 48 hr and late centrifuged (40°C, 8,000 rpm, for 10 min). Then, the resultant precipitation was dissolved in dH2O and the EPS was defined.

| Quantification
Five per cent phenol solution was prepared in water by dissolving 5 g of fresh phenol in dH2O and the volume was made to 100 ml.
For the calibration purpose, 1 mg/ml glucose solution and six different standards of glucose were prepared. Four hundred microliters of sample and 400 µl of 5% phenol solution in water were mixed in the glass test tube. A control sample was also prepared in which 400 µl of dH2O and 400 µl of 5% phenol solution in water were used. Then, 2 ml of concentrated sulfuric acid was added to the solution into the tube and left for 10 min. Then, the mixture was shaken and again left for 10 min at 30°C. Then, the samples were run at 490 nm in quartz on 752 UV-Vis spectrophotometer cuvettes, and the reading was recorded and compared with control. The amount of EPS (mg) was calculated using glucose calibration line (Feldmane, Semjonovs, & Ciprovica, 2013).

| Technological screening
The milk (12% reconstituted skim milk) was fermented with identified EPS-producing Lb. delbrueckii ssp. bulgaricus strains at 37°C till curd is settled down. Technological parameters such as curdling time, titratable acidity, flavor, and texture and body were evaluated.
Titration method was used to evaluate titratable acidity from fermented milk products, and sensory method was used to estimate the curdling time, body and texture, and flavor.

| Preparation of yogurt by selected EPSproducing strains of Lb. delbrueckii ssp. bulgaricus by using cow milk
Fresh cow milk was obtained from National Agriculture Research Center (NARC), Islamabad. The raw milk contains total solids 12.5%, total protein 3.30%, lactose 4.6%, fat 3.8%, pH 6.65, and titratable acidity 0.14. The raw milk was heated up to 40°C, and their solid contents were increased up to 15% by adding skim milk powder. Then, milk was heated at 90°C for 30 min and cooled to 42°C, and then, milk was divided into four equal portions 200 ml for each and incubated with EPS-producing strains that were identified in our research and incubated with four different combinations that were as follows: Each portion of cow milk was inoculated with 2% (v/v) starter cultures; then, milk was transferred into 200 g plastic cups and incubated at 42°C for 4-5 hr until coagulation, and then, cups were cooled down at 4°C and stored for 21 days. Then, the yogurt was analyzed experimentally on weekly basis as chemical or organoleptical.
The experiment was repeated three times in duplicate.

| Chemical analysis
The pH of yogurt sample was measured by pH meter and combined glass electrode. EPS contents from yogurt sample were determined by the method described by Dubois, Gilles, Hamilton, Rebers, and Smith (1956). One volume of fermented milk was mixed with one volume of 20% (wt/vol) trichloroacetic acid (TCA), and then heated at 100°C for 5 min and centrifuged at 3,500 g at 20°C for 10 min. The supernatant was removed after centrifugation and 0.5 volumes of 10% TCA was added and again centrifuged. Aqueous phases were pooled and dialyzed at 4°C against deionized water for 4 days. Then, EPS concentration in the suspension was quantified by phenol-sulfuric acid method and was expressed as glucose equivalent with glucose as a standard.

| Apparent viscosity
Apparent viscosities of yogurt were measured according to method described by Shihata and Shah (2002). The apparent viscosity was measured on cup at 20°C with a Brookfield viscometer after 1, 7, 14, and 21 days of storage. The spindle used (LV-SC4-34 spindle at 4 rpm) in 150 g of yogurt was allowed to rotate for 1 min at 20°C.

| Syneresis
Susceptibility of yogurt to syneresis was determined by centrifuging 20 g of sample at 500 rpm for 5 min, weighing the supernatant, and then measuring the amount of supernatant recovered. Percent syneresis was calculated as:

| Sensory evaluation
Twenty trained panelists (14 women and six men, aged  were asked to evaluate the sensory attributes of yogurt. The ratings were presented on a 9-point hedonic scale ranging from 9 ("like extremely") to 1 ("dislike extremely"). Yogurt sensory parameters were evaluated by thickness, smoothness, fermented odor, finished flavor, and taste quality. The yogurts were served to panelists after the cooling process. Result was given on averages of the three trials for each type of yogurt (Sahan, Yasar, & Hayaloglu, 2008).

| Statistical analysis
Completely randomized design was used for statistical analysis for experimental data. Statistical analyses were performed using SPSS 14.0 software (SPSS Inc.; Chicago, IL, USA). ANOVA followed by Tukey's test was used for statistical differences with a level of significance = 0.05 (Han et al., 2016).

| Isolation of EPS-producing Lb. delbrueckii ssp. bulgaricus strains
Exopolysaccharide-producing bacterial culture produced mucoid colonies on milk agar and MRS agar plates. Morphological examination of these colonies was done by Gram staining, and based on their morphology, colonies containing long rods or rods in chains were picked and repetitive streaking method was followed to purify these colonies using the same method for isolation of lactic acid bacteria described by Behare et al. (2010). culture-independent analysis of the fermented sample. In the past, Suhartatik, Cahyanto, Rahardjo, Miyashita, and Rahayu (2014) reported the identification of different strains by PCR amplification using similar method.

| Technological screening
For the evaluation of technological properties, all the selected EPSproducing Lb. delbrueckii ssp. bulgaricus strains were further tested for their curdling time, acidity, and body and texture. Technological properties of selected strains are summarized in Table 2. It was observed that the four strains L1, L3, L5, and L7 showed better results in flavor, and body and texture of final product as compared to other tested strains. There was inverse relationship between curdling time and titratable acidity but no clear-cut relationship with EPS production because EPS-producing genes were located on chromosomes, so EPS production varies from strain to strain.
The technological parameters that were greatly affected by EPS production were body and texture, and production of EPS improved these parameters.

| Preparation of yogurt by selected EPSproducing strains of Lb. delbrueckii ssp. bulgaricus by using cow milk
The chemical composition of cow's fermented product made with EPS-and non-EPS-producing starter culture during storage of 21 days at 4°C is presented in Table 3. There is a significant dif- by Han et al. (2016) and proved that yogurt made with EPS-producing strains showed more viscosity as compared to yogurt made with non-EPS-producing strains and little increase in EPS can affect more viscosity and adhesiveness. The viscosity of yogurt sample increased due to the presence of EPS-producing strains in starter culture as reported by Gürsoy et al. (2010).
The firmness of yogurt made with non-EPS-producing starter culture was higher in control sample as compared to yogurt made with ropy EPS-producing starter cultures. The highest value of firmness was found after 1 day of storage of fresh control sample treatment, and there was a significant difference (p < 0.05) between readings of different days of storage. Similar results were reported by Gürsoy et al. (2010) that the gel firmness was not affected by EPS-producing starter culture. Results also showed that yogurt made with EPS-producing starter culture showed lower syneresis as compared to yogurt made with non-EPS-producing starter culture (control). This result showed that syneresis of fermented milk product with EPS-producing starter culture depends upon the ability of EPS to bind water and that ability is affected by its type and concentration in the product and their interaction and distribution with protein network. Furthermore, longer fermentation time of cow milk allowed more structural rearrangement due to this weak structure formed and spontaneous syneresis increase. The collected data showed that sensory quality acceptance and the visual appearance of yogurt made with EPS-producing strains were same as those of yogurt produced by non-EPS-producing strains and the gels were smooth and free of syneresis. The yogurt made with non-EPS-producing starter culture (control) had significantly (p < 0.05) lower rating for texture and body, color, flavor, and appearance and overall score, due to whey off on the fermented milk surface (Table 4). Moreover, rich mouthfeel and good acid taste were observed in treatments B, C, and D, and flavor was also good after 14 days of storage period, but the appearance and acceptability decreased due to whey off on the fermented milk surface.
The overall acceptability scores of the yogurt made by EPS-producing starter culture were higher and acceptable in treatment C as compared to control treatment. The flavor of yogurt was more preferred by panel of consumer that was made with non-EPS-producing starter culture as the score was higher (p < 0.05). This difference is due to the presence of more acetaldehyde contents in yogurt that was made with non-EPS-producing starter culture. an innovative idea to replace artificial stabilizers such as gums, gelatin, pectin with these natural EPS-producing bacterial strains.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.