The Tolerance of Lactobacillus paracasei and Lactobacillus curvatus Originated from Bovine Colostrum towards Acidity and Bile Salts as a Probiotics Candidate

The study conducted to obtain Lactobacillus species from bovine colostrum as probiotic candidate that have resistant character towards acidity and bile salt condition. Research done experimentally with Factorial Completely Randomized Design Pattern. The results were analyzed using Analysis of Variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). Results showed that L. paracasei and L. curvatus have tolerance of pH 2 with colony population by 0.07×10 2 CFU/mL. L. paracasei and L. curvatus also have tolerance as well towards 0.3% of bile salt concentration with colony population by 4.05×10 10 CFU/mL and 0.5% of bile salt concentration with colony population by 4.02×10 10 CFU/ml. L. paracasei and L. curvatus have character as probiotic because grew well at high acidity and bile salts condition.


Probiotic
bacteria are non-pathogenic microorganisms that can control microbial populations in the human digestive system and has a benefit to its host.The competition mechanisms that can control population of pathogenic bacteria in the digestive system provided by probiotic bacteria (De Smet et al., 1995;Bezkorovainy, 2006).Main source of probiotics can be obtained from wide variety of food products made from milk, such as colostrum, cottage cheese, yogurt, kefir, or milk powder enriched with probiotics (Gómez Zavaglia et al., 1998).
Colostrum is a yellow-colored solution produced by the mammary glands of cattle between 24-96 h after birth.Colostrum will not produce after 4-5 days of birth because it will turn into milk (Kusumawati, 2002).Colostrum contains bifigenic factor proved to be Nacetylglucosamine that can support the growth of lactic acid bacteria (Lactobacillus sp., Lactococcus sp., Bifidobacteria) (Kashket, 1987;Salminem et al., 2004).
Lactobacillus sp. is one of the excellent bacteria that often used as a probiotic culture.These bacteria have the criteria for probiotic bacteria such as viability at high acidity and bile salt tolerance (Kusumawati, 2002).The study aimed to isolated Lactobacillus sp.originated from bovine colostrum that have character of acidity and bile salts tolerance so it can be used as probiotic bacteria.

Method:
Tolerance of acidity test method: L. paracasei and L. curvatus incubated at NB at 30°C for 24 h.Set the acidity level pH 2, 4, 6 by 20 mL of NB using 1% acetic acid and 1% NaOH as a buffer.Each treatment inoculated with 10% starter during the logarithmic phase (log) into acidified NB.Control (blank) was prepared using NB with pH that set without addition of starter.Treatment incubated for 6 hours.At pH 6 condition, we do eight dilution series (10 -8 ).Total Plate Count (TPC) method used to count the population of bacteria with NA after incubation for 24 hour (Hartati, 2007).
Tolerance of Bile Salts Test Method: L. paracasei and L. curvatus incubated at NB at 30°C for 24 h.Set the bile salt level of 0.3 and 0.5% by NA using (Oxoid) bile salt.One mL of 10 serial dilutions (10 -10 ) liquid culture grown on plates with pour plate method.Total Plate Count (TPC) method used to count the population of the bacteria with NA after incubation for 24 h (Buntin et al., 2008).

RESULTS AND DISCUSSION
Tolerance of acidity: Probiotic Bacteria generally have high tolerance to acidic pH in the range of 2-4.This is because the stomach has very low pH conditions ranging from 2-4 (Gómez Zavaglia et al., 1998).Results of Analysis of Variance (ANOVA) showed that the pH gave significant effect on colony growth resistance of each test bacteria (L.paracasei and L. curvatus), but no interaction between pH and the type of bacteria.The lower levels of pH, the number of bacterial population growth will decrease more.Results of the Analysis of Variance (ANOVA) was performed Duncan's Multiple Range Test which can be shown in Table 1.
At pH two, the number of bacteria population was 0.07×10 2 CFU/mL, which indicates that the grown bacteria have tolerance towards low pH.At pH four, the average population of bacteria was 0.498×10 2 CFU/mL.Both bacteria still grown at pH 2 and pH 4, which indicates that the bacteria can be candidate as probiotic bacteria (Hardianingsih et al., 2006).
Condition of digestive tract especially the human being was in the low pH range.Stress happens to the bacterial cell when it enters the digestive tract and exposed to acid.Potential probiotic bacteria must be resistant to the low pH conditions of the stomach in the range of 2-4 (Hardianingsih et al., 2006).Kashket (1987) and Kimoto et al. (2004) states that Lactobacillus is bacteria that resistant towards acidic conditions.
Acidic conditions on the media obtained by adding acetic acid in liquid medium (Nutrient Broth).Exposure to high acidic conditions may result damage to the cell membrane and the release of intracellular components that are capable of causing bacterial cell death.Bacteria that are tolerance towards low pH has a resistance to membrane damage due to decrease of extracellular pH.Lactobacillus is lactic acid bacteria and probiotic bacteria that can live in wide pH range.The main defense of the bacteria towards the environment is cell membrane structure consisting of two layers of fat (lipid bilayer).Hartati (2007) states that L. casei and L. paracasei can grow at extreme low pH conditions that making it suitable to be candidate as probiotic bacteria.
The difference in susceptibility to the cytoplasmic membrane of bacteria will determine how big the bacterial tolerance to low pH.To survive in an acidic environment, Lactobacillus must maintain intracellular pH higher than the extracellular pH.Intracellular pH of Lactic acid bacteria such as Lactobacillus could dynamically changes along with decrease in extracellular pH (Drouault et al., 1999;Hill, 1995).Drouault et al., (1999) suggested that the enzyme might also affect the growth of Lactobacillus as lactic acid In accordance with Bezkorovainy (2006), the effect of probiotic bacteria in the intestinal environment can be occur when the population of the bacteria reach at least 10 9 -10 11 CFU/mL at low pH.L. paracasei and L. curvatus obtained from bovine colostrum has the population below the standard for probiotic acitivities.Acclimatization or optimization needed if L. paracasei and L. curvatus originated from bovine colostrum will used as probiotic, so that the population of bacteria can meet the standards.

Tolerance of bile salts:
The test performed by growing L. paracasei and L. curvatus isolates in NA that added by bile salts with concentration of 0.3% and 0.5%.Concentration of 0.3 and 0.5% selected as the concentration equivalent to the concentration of bile salts in duodenum physiologically (Gómez Zavaglia et al., 1998).According to analysis of variance (ANOVA) at concentration of 0.3% bile salts and 0.5% there is no significance difference between the growth of L. paracasei and L. curvatus at the bile salt concentration of 0.3% and 0.5% so it does not proceed with the test Duncan's Multiple Range.At bile salt concentration of 0.3% and 0.5%, L. paracasei and L. curvatus grew optimally with not significantly different amount.The results of the average population of L. paracasei and L. curvatus in the bile salt concentration of 0.3 and 0.5% can be seen in Table 2.
The average population of L. paracasei at concentrations of 0.3% bile salts was 3.87×10 10 CFU/mL and at concentrations of 0.5% bile salts were 4.76 x 10 10 CFU/mL.Meanwhile the growth of the bacterial population of L. curvatus at concentration of 0.3% bile salts was 4.24×10 10 CFU/mL and at Results showed that both bacteria grew well with the same relative population in the media with addition of bile salts concentrations of 0.3% and 0.5%.This shows that both bacteria have a strong character as a probiotic candidate because it can grow optimally at each concentration of bile salts tested.In order to survive and grow in the gastrointestinal tract, Lactobacillus as a probiotic culture must be able to pass various environmental conditions such as high concentration of bile salts in the upper intestinal tract where bile secreted into the intestine.Bile is a mixture of bile acids, cholesterol, fatty acids, phospholipids, bile pigments and lipolytic enzymes that activated by surface-active characteristics of bile (Xiao et al., 2003).Bile combination is bactericidal for commensal microorganisms in the human body except for the few inhabitants of the gut that are resistant to bile salts (Kimoto et al., 2004).Both bacteria tested at bile salts concentrations of 0.3 and 0.5% that is the critical concentration and high enough bile salts amount to test the isolates that were resistant to bile salts (Xiao et al., 2003).
Good tolerance to bile salts is presumably due to the role of polysaccharides as one a constituent of the cell wall of gram-positive bacteria.Lactobacillus known as the group of lactic acid bacteria has a resistance to mixed bile salts (Saarela et al., 2000;Salminen et al., 1999).According Bezkorovainy (2006) liquid bile salts are surface-active compounds that can penetrate and react with the cytoplasmic side of the membrane which are lipophilic which can cause changes in bacterial membrane structural damage.Lactobacilli have bile salt hydrolase enzyme activity to hydrolyze bile salts.This enzyme is able to alter the physical and chemical capabilities possessed by bile salts, so it is not toxic to the Lactobacillus.This is what allows some Lactobacillus isolates resistant to bile salts circumstances (Fig. 1 and 2).

CONCLUSION
Based on the results it can be concluded that L. paracasei and L. curvatus have ability to tolerate high aciditiy environment and high concentration of bile salts, so that can be candidate as probiotic.

Table 1 :
Duncan's multiple range test (level of 5%) acidity into bacteria population Lactobacillus is able to maintain the acidity of the cytoplasm so that proteins and enzymes inside the cell can still work optimally.Based on the results, L. paracasei and L. curvatus potential as probiotic candidates because the ability to grow at acidic pH range.