Sample preparation:

 

• 0.010 g of culture inoculated in 100 ml of sterilizaed MRS broth (pH 5.4).

 

• Incubate at 40ºC for 2 h prior to experiment.

 

Experimental set up:

 

Experiment 1 – Effect of processing time on the survival and growth of LAB at 50% amplitude.

Time of exposure – 10, 20 and 30 min

 

Experiment 2 – Effect of processing time on the survival and growth of LAB at 70% amplitude.

Time of exposure – 7, 14 and 21 min

 

Experiment 3 – Effect of output power on the survival and growth of LAB.

Time of exposure – 14 min

Amplitude: 50%, 70% and 90%

 

Experiment 4 – Effect of varying output power (W) and treatment time (min) while keeping the same total energy output (W·min·cm^-3 ).

 

Procedure:

 

• Pour out 10 ml of inoculated sample and set aside to be used as control.

 

• Process the inoculated sample according to the processing conditions.

 

• Remove 20 ml of sample for evaluation.

 

Evaluation of samples:

 

Plate count of B. longum after ultrasound processing

 

• Remove 20 ml of processed sample.

 

• Perform decimal dilution to achieve dilutions of 10^-1 to 10^-6.

 

• Transfer 1 ml of each dilution into each plate. Triplicates are performed for each dilution.

 

• Incubate for 48 h at 37ºC and count the colonies.

 

Measure the growth of  B. longum using spectrophotometric method at 600 nm

 

• Measurements are taken before and after 5 h of incubation at 37℃.

 

Acid production of B. longum

 

• pH measurements are performed before and after 5 h of incubation at 37℃.