In-vivo evaluation of the anti-diarrheal effect of Lactococcus lactis subspecies lactis and Lactococcus piscium isolated from yogurt

Lactobacillus and Lactococcus species found in the yogurt of different sources are most widely assayed and used all over the world as a probiotic agent. This study aimed to isolate and identify novel probiotic agents with therapeutic value against diarrhea. Initially, the probiotic properties of the isolated lactic acid bacteria from the yogurt samples of the Chittagong division, Bangladesh, were evaluated. All probiotic candidates inhibited the growth of selected pathogens, including Escheriachia coli, Serratia sp. Salmonella paratyphi, Streptococcus Group-B, Staphylococcus aureus, Haemophillus influenzae, Bacillus subtillis, and Klebsiella pneumoniae. Lactococcus lactis subsp lactis were found most useful in inhibiting all the selected pathogens. When the probiotics were applied against castor oil-induced diarrhea in the rat model, Lactococcus lactis subspecies lactis and Lactococcus piscium were found significantly effective relative to the controls indicating their potentiality as an alternative therapeutic against diarrhea. Highlights Lactococcus lactis subspecies lactis and Lactococcus piscium has shown potentiality to be a therapeutic agent against castor-oil induced diarrhea in an animal model. Lactococcus lactis subspecies lactis and Lactococcus piscium inhibited the growth of specified pathogens.


Introduction
Probiotic bacteria obtained with food are frequently claimed to improve human health. From the view of the clinical field, probiotics are very beneficial for their unique therapeutic properties and inducing responsive actions. In vitro study revealed that the gut Lactic acid bacteria influence immunomodulation, induce the production of anti-inflammatory cytokines, and thus prevent the suspension of five isolates and incubated at 27 ˚C, 37 ˚C and 45 ˚C temperature for 24-48 hours.
Using a spectrophotometer at 560 nm, the turbidity was measured and filtered with the help of the Whatman filter paper (Whatman International Ltd. England). Measuring pH, the culture filtrate (Hanna Instrument Ltd. U.K.) was applied for antimicrobial activity against the respective pathogenic bacteria by the agar well diffusion method.

Bile salt tolerance of the isolates
With different concentrations (i.e., 1.0%, 2.0%, and 3.0%) of bile salt, MRS broth was prepared and dispensed at 10 ml per tube with the selected isolates. The incubation condition was 37 ± 1 ˚C for 24-48 hours. Using the pour plate method, 100 μ l of culture from each concentration was grown in agar medium at 37 ˚C for 24 hours for comparative growth.

Activities of selected lactic acid against specified pathogens
The procedure for observing the activity of selected lactic acid bacteria against pathogens developed previously. Briefly, an overnight grown pathogenic bacteria culture was suspended in 2 ml of sterilized saline water and mixed thoroughly. A total of seven pathogenic microorganisms were used in this method, namely Bacillus subtilis, Staphylococcus aureus. Streptococcus Group-B, Escherichia coli, Serratia sp., Klebsiella pneumonia, and Haemophillus influenzae. Growth inhibition was done in Mueller Hinton by the Well diffusion method, and media were seeded uniformly with 2.7×10 3 cells per ml of test organisms and incubated at 37 o C for 24 hours. The anti-bacterial activity of the test agent was determined by measuring the zone of inhibition expressed in mm in diameter.

Assay of Antibiotic Sensitivity Pattern
To determine the antibiotic sensitivity pattern, the disk diffusion method was followed. In this method, Mueller Hinton plates were prepared and swabbed with the suspension of selected isolates using a sterile cotton bud. The antibiotic disks were placed on the surface of the plate, maintaining equal distance and placed at 4˚C for 1-2 hours for proper diffusion of antibiotics followed by incubation for 18-24 hours at 37˚C. The zone of inhibition measured by the zone of diameter for antibiotic sensitivity or resistance [28].

In-vitro testing for the anti-diarrheal effect of selected isolates
The in vivo anti-diarrhoeal activity of the Castor oil-induced diarrheal model was performed by [29] suggested method. Thirty-five Wistar rats were randomly divided into seven equal groups (n=5), Control group, positive control group, and five treated groups. Control group were received only distilled water 2 ml/rat, the positive control group received Loperamide (Opsonin, Bangladesh) 4.17 mg/kg as standard and treated groups received selected isolates or probiotics at the dose of fecal matters. Diarrhea was induced in rats by oral administration of castor oil (2.0 ml/rat). The antidiarrheal activity was determined by giving probiotics and drugs orally 1 hour before the administration of a standard dose of 2.0 ml of castor oil. The number of both hard and soft pellets was enumerated at every hour over 6 hours period for each rat. Diarrhea was confirmed as the presence of stool with a fluid material that stained the paper placed beneath the cages.

Statistical analysis
All the statistical values of anti-diarrheal, tests were reported as mean + SEM (Standard error of the mean). Statistical differences between the mean of the various groups were analyzed using the Student's "t" test. Probability (p) value of 0.05 or 0.01 was considered significant. All the graphical presentations and statistical calculations were prepared using "Microsoft Excel-2007". Mean values were considered significantly different if P< 0.05, 0.01.

Bile salt tolerance
As probiotic organisms must tolerate varying concentrations of bile salt in the intestinal tract, the isolates were tested for their sensitivity to different concentrations of bile salt (i.e., 1%, 2%, and 3%).

Assay of Antibiotic Susceptibility pattern
The antibiotic sensitivity pattern of the selected isolates was also determined to investigate the severe effect of any antibiotics against them.  The measured diameter of inhibition zones accomplished that all the isolates have anti-bacterial effects against the pathogenic microorganisms ( Figure 1). The Lactococcus species was evaluated in castor oil-induced diarrheal rats as compared to normal rats. The feces at sixth hours for Lactococcus species treated group at 10 6 CFU/ML were significantly (p<0.05, p<0.01) decreased as compared to the control group (Table 4).

Discussion
Acute diarrhea is one of the significant reasons for childhood morbidity as well as a load on society, which comes with an economic and emotional burden for the families of patients.  (Table 2).
It has been reported that most Lactococcus species exhibit intrinsic resistance to trimethoprim, metronidazole, and cefoxitin, and the aminoglycosides kanamycin and gentamicin. Also, L. lactis found to carry antibiotic resistance genes erm(B), tet(M), tet(S), and dfr(A) [41]. In a different study, three multi-drug resistance genes, namely lmr(A), lmr(D), and lmr(P), were found in L. lactis [42].
Previously, L. raffinolactis found to be not resistant to Ciprofloxacin, Erythromycin, and Gentamicin, Though all of our selected isolates were susceptible to some antibiotics resistance to other, three isolates of genus Lactococcus showed the lesser difference between the percentage of antibiotics that they were resistant and the number of antibiotics that they were susceptible ( Figure 2 S  e  l  e  c  t  i  o  n  o  f  p  o  t  e  n  t  i  a  l  p  r  o  b  i  o  t  i  c  l  a  c  t  i  c  a  c  i  d  b  a  c  t  e  r  i  a  f  r  o  m  f  e  r  m  e  n  t  e  d  o  l  i  v  e  s  b  y  i  n  v  i  t  r  o  t  e  s  t  s  .   F  o  o  d  M  i  c  r  o  b  i  o  l  o  g  y  ,  2  0  1  3  .   3  3   (  2  )  :  p  .  2  8  2  -2  9  1  .   2  7  .  S  a  b  i  r  ,  F  .  ,  e  t  a  l  .  ,   A  s  s  e  s  s  m  e  n  t  o  f  P  o  t  e  n  t  i  a  l  P  r  o  b  i  o  t  i  c  P  r  o  p  e  r  t  i  e  s  o  f  L  a  c  t  o  b  a  c  i  l  l  u  s  s  p  p  .  ,  L  a  c  t  o  c  o  c  c  u  s  s  p  p  .  ,  a  n  d  P  e  d  i  o  c  o  c  c  u  s  s  p  p  .  S  t  r  a  i  n  s  I  s  o  l  a  t  e  d  f  r  o  m  K  e  f  i  r  .   J  o  u  r  n  a  l  o  f  F  o  o  d  S  c  i  e  n