Low-fat Chicken Biryani Rice

Chicken Biryani Rice is one of the Indian traditional cuisine and in this recipe, i use a new approach that will eventually turn it into a low fat dish that suit todays healthy lifestyle.

Recipe:
Ingredient:
10 Chicken pieces
Ground coriander seeds
Grount cloves
Grated garlic
Sliced onion
8 curry leaves
1.5 cups of rice
1 tomato-sliced
1 tbsp coconut shred
1 tbsp milk
1.5 cups of warm water
1 tsp of turmeric powder
half tsp of cumin
1 tsp of chili powder

Marinate the chicken pieces with ground coriander seeds, salt and ground cloves.
Wash and drain 1.5 cups of short grain rice.
Saute the grated garlic, sliced onion and curry leaves. Add chili powder.
Add chicken to it and fry for 15 minutes.
Add rice and stir.
Add turmeric, cumin, and soy sauce to your liking.
Meanwhile, add milk and coconut.
pour warm water and stir. Transfer to rice cooker.
Serve hot.

My Thesis: REFERENCES

CHAPTER 6

REFERENCES

 

 

1. Alvarez-Olmos, M.A., and Oberhelman, R.A., 2001. Probiotic Agents and Infectious Diseases: A Modern Perspective on a Traditional Therapy. Clinical Infectious Diseases, 32, 1567–1576.
   
2. Arvola, T., Laiho, K., Torkkeli, S., Mykkanen, H., Salminen, S., Maunula, L., and Isolauri, E., 1999. Prophylactic Lactobacillus GG reduces Antibiotic-Associated Diarrhea in Children with Respiratory Infections: a Randomized Study. Pediatrics, 104(5),1-4.
   
3. Berrada, N., Lemeland, J.F., Laroche, G., Thouvenet, P., and Piaia, M., 1991. Bifidobacterium from Fermented Milks: Survival during Gastric Transit. Journal of Dairy Science, 74, 409-413.
   
4. Bezkorovainy, A., 2001. Probiotics: determinants of survival and growth in the gut. American Journal of Clinical Nutrition, 73, 399S–405S
   
5. Bibiloni R, Perez PF, Garrote GL et al. Surface characterization and adhesive properties of bifidobacteria. Methods in Enzymology 2001; 336: 411–427.
   
6. Brown, A.C., and Valiere, A., 2004. Probiotics and Medical Nutrition Therapy. Nutritional Clinical Care, 7(2), 56–68.
   
7. Bruno, F.A., and Shah, N.P., 2003. Viability of two freeze-dried strains of Bifidobacterium and of commercial preparations at various temperatures during prolonged storage. Journal of food science, 68 (7), 2336-2339.
   
8. Campbell, N.A., 1996. Biology, 4th edn. CA: Benjamin/Cummings.
   
9. Charalampopoulos, D., Pandiella, S.S., Webb. C., 2003. Evaluation of the effect of malt, wheat and barley extracts on the viability of potentially probiotic lactic acid bacteria under acidic conditions. International Journal of Food Microbiology, 82,133-141.
   
10. Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K., 1998. Development and Application of An In Vitro Methodology to Determine the Transit Tolerance of Potentially Probiotic Lactobacillus and Bifidobacterium Species in the Upper Human Gastrointestinal Tract. Journal of Applied Microbiology, 84, 759–768.
    
11. Chou, L.S., and Weimer, B., 1999. Isolation and Characterization of Acid- and Bile-Tolerant Isolates from Strains of Lactobacillus acidophilus. Journal of Dairy Science, 82, 23–31.
    
12. Chung, H.S., Kim, Y.B., Chun, S.L., and Ji, G.E., 1999. Screening and Selection of Acid and Bile Resistant Bifidobacteria. International Journal of Food Microbiology, 47, 25–32.
    
13. Collado, M.C., and Sanz, Y., 2006. Method for Direct Selection of Potentially Probiotic Bifidobacterium Strains from Human Feces Based on their Acid-Adaptation Ability. Journal of Microbiological Methods, 66, 560–563.
    
14. Corcoran, B.M., Stanton, C., Fitzgerald, G.F., and Ross, R.P., 2005. Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars. Applied And Environmental Microbiology, 3060–3067.
    
15. Cotter, P.D., and Colin, H., 2003. Surviving the acid test: responses of Gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews, 429–453.
    
16. Cruchet, S., Obregon, M.C., Salazar, G., Diaz, E., and Gotteland, M., 2003. Effect of the ingestion of a dietary product containing Lactobacillus johnsonii La 1 on Helicobacter pylori colonization in children. Nutrition, 19,716–721.
    
17. D'souza, A. L., Rajkumar, C., Cooke, J., and Bulpitt, C. J., 2002. Probiotics in prevention of antibiotic-associated diarrhoea: Meta analysis. British Medical Journal, 324, 1361– 1364.
    
18. Dave, R.I., Shah, N.P., 1997. Viability of yogurt and probiotic bacteria in yogurts made from commercial starter cultures. Int Dairy J , 7, 31–41.
    
19. Dunne, C., O'Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O'Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G., Daly, C., Kiely, B., O'Sullivan, G.C., Shanahan, F., and Collins, J.K., 2001. In Vitro Selection Criteria for Probiotic Bacteria of Human Origin: Correlation with In Vivo Findings. American Journal of Clinical Nutrition, 73, 386S–92S.
    
20. Egawa, H., Yonemochi, E., and Terada, K., 2005. Investigation of optimal manufacturing process for freeze- formulations: Observation of frozen solutions by low temperature X-ray diffraction measurements. Thermochimica Acta , 431, 127-132.
    
21. Fernandez-Banares, F., 2006. Nutritional Care of the Patient with Constipation. Best Practice and Research Clinical Gastroenterology, 3(20), 575–587.
    
22. Fioramonti, J., Theodorou, V., and Bueno, L., 2003. Probiotics: What Are They? What Are Their Effects on Gut Physiology? Best Practice and Research Clinical Gastroenterology, 17(5), 711–724.
    
23. Food and Agriculture Organization (FAO) of the United Nations and World Health Organization (WHO). Guidelines for the Evaluation of Probiotics in Food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. Accessed at http://www.who.int/foodsafety/fs_management/en/ probiotic_guidelines.pdf on December 7, 2006.
    
24. Fooks, L.J., Fuller, R., and Gibson, G.R., 1999. Prebiotics, Probiotics and Human Gut Microbiology. International Dairy Journal, 9, 53-61.
    
25. Fooks, L.T., and Gibson, G.R., 2002. Probiotics as modulators of the gut flora. British Journal of Nutrition, 88 (Suppl.1), S39–S49.
    
26. Forestier, C., De Champs, C., Vatoux, C., and Joly, B., 2001. Probiotic activities of Lactobacillus casei rhamnosus: in vitro adherence to intestinal cells and antimicrobial properties. Research in Microbiology, 152,167–173.
    
27. Gill, H.S., 2003. Probiotics to enhance anti-infective defences in the gastrointestinal tract. Best Practice & Research Clinical Gastroenterology, 17(5), 755–773.
    
28. Gismondo, M.R., Drago, L., and Lombardi, A., 1999. Review of Probiotics Available to Modify Gastrointestinal Flora. International Journal of Antimicrobial Agents, 287–292.
    
29. Gopal, P.K., Prasad, J., Smart, J., and Gill, H.S., 2001. In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli. International Journal of Food Microbiology, 67, 207–216.
    
30. Guarner, F., and Malagelada, J.R., 2003, Gut Flora in Health and Disease. Lancet, 361, 512–519.
    
31. Holzapfel, W.H., Haberer, P., Geisen, R., Bjorkroth, J., and Schillinger, U., 2001. Taxonomy and Important Features of Probiotic Microorganisms in Food and Nutrition. American Journal of Clinical Nutrition, 73(2), 365S-373s.
    
32. Holzapfel, W.H., Haberer, P., Snel, J., and Schillinger, U., 1998. Overview of Gut Flora and Probiotics. International Journal of Food Microbiology, 41, 85–101.
    
33. Huang, Y., and Adams, M.C., 2001. In Vitro Assessment of the Upper Gastrointestinal Tolerance of Potential Probiotic Dairy Propionibacteria. International Journal of Food Microbiology, 91, 253– 260.
    
34. Hutkins, R.W., and Nannen, N.L., 1993. pH Homeostasis in Lactlc Acld Bacterial. J Dairy Sci 76, 2354-2365.
    
35. Kailasapathy, K., and CHIN, J., 2000. Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunology and Cell Biology, 78, 80–88.
    
36. Kailasapathy, K., and Chin, J., 2000. Survival and Therapeutic Potential of Probiotic Organisms with Reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunology Cell Biology, 78, 80–8.
    
37. Klein, G., Pack, A., Bonaparte, C., and Reuter, G., 1998. Taxonomy and Physiology of Probiotic Lactic Acid Bacteria. International Journal of Food Microbiology, 41, 103–125.
    
38. Klijn, A., Mercenier, A., and Arigoni, F., 2005. Lessons from the Genomes of Bifidobacteria. Federal of European Microbiological Societies Microbiology Reviews, 29, 491–509.
    
39. Koebnick, C., Wagner, I., Leitzmann, P., Stern, U., and Zunft, H.J., 2003. Probiotic Beverage Containing Lactobacillus casei shirota Improves Gastrointestinal Symptoms in Patients with Chronic Constipation. Canadian Journal of Gastroenterology, 17, 655–659.
    
40. Kotowska, M., Albrecht, P., and Szajewska, H., 2005. Saccharomyces boulardii in the Prevention of Antibiotic-Associated Diarrhea in Children: a Randomized Double-Blind Placebo-Controlled Trial. Alimentary Pharmacology and Therapeutics ,21,583-590.
    
41. Lapierre, L., Undeland, P., and Cox, L.J., 1992. Lithium Chloride-Sodium Propionate Agar for the Enumeration of Bifidobacteria in Fermented Dairy Products. Journal of Dairy Science, 75, 1192-1196.
    
42. Lee, Y.K., Lim, C.Y., Teng, W.L., Ouwehand, A.C., Tuomola, E.M., and Salminen, S., 2000. Quantitative Approach in the Study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria. Applied and Environmental Microbiology, Sept, 3692–3697.
    
43. Lin, W.H., Hwang, C.F., and Chen, L.W., 2006. Viable Counts, Characteristic Evaluation for Commercial Lactic Acid Bacteria Products. Food Microbiology, 23, 74-86.
    
44. Ling, M.T., and Shah, N.P., 2005. Acid and bile tolerance and cholesterol removal ability of Lactobacilli strains. J. Dairy Sci., 88, 55-66.
    
45. Liong, M.T., and Shah, N.P., 2005. Acid and Bile tolerance and Cholesterol Removal Ability of Lactobacilli Strains. Journal of Dairy Science, 88, 55-66.
    
46. Marteau, P., 2003. Basic aspects and pharmacology of probiotics: an overview of pharmacokinetics, mechanisms of action and side-effects. Best Practice & Research Clinical Gastroenterology, 17(5), 725–740.
    
47. Marteau, P., Cuillerier, E., Meance, S., Gerhardt, M.F., Myara, A., Bouvier, M., Bouley, C., Tondu, F., Bommelaer, G., and Grimaud, J.C., 2002. Bifidobacterium animalis strain DN-173 010 shortens the colonic transit time in healthy women: a double-blind, randomised, controlled study. Aliment Pharmacology and Therapeutics, 16,587–593.
    
48. Masco, L., Huys, G., Brandt, E.D., Temmermen, R., and Swings, T., 2005. Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria. International Journal of Food Microbiology, 102, 221– 230.
    
49. Matsumoto, M., Ohishi, H., and Benno, Y., 2004. H⁺-ATPase Activity in Bifidobacterium with Special Reference to Acid Tolerance. International Journal of Food Microbiology, 93, 109– 113.
    
50. Mcfarland, L.V., 2005. Meta-Analysis of Probiotics for the Prevention of traveler's diarrhea. The American Journal of Gastroenterology,
    
51. McFarland, L.V., and Elmer, G.W., 1997. Pharmaceutical Probiotics for the Treatment of Anaerobic and Other Infections. Anaerobe, 3, 73–78.
    
52. Mishra, V., and Prasad, D.N., 2005. Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. International Journal of Food Microbiology, 103, 109– 115.
    
53. Morgan, C.A., Herman, H., White, P.A., Vesey, G., 2006. Preservation of microorganisms by drying; A review. Journal of Microbiological Methods, 66, 183–193.
    
54. Nannen, N.L., and Hutkins, R.W., 1991. Intracellular pH effects in lactic acid bacteria. J Dairy Sci. 74, 741-746.
    
55. Nomoto, K., 2005. Prevention of Infections by Probiotics. Journal of Bioscience and Bioengineering, 100 (6), 583–592.
    
56. O'Sullivan, G.C., Kelly, P., O'Halloran, S., Collins, C., Collins, J.K., Dunne, C., and Shanahan, F., 2005. Probiotics: An Emerging Therapy. Current Pharmaceutical Design, 11, 3-10.
    
57. Ouwehand, A.C., Lagstrom, H., Soumalainen, T., and Salminen, S., 2002. Effect of Probiotics on Constipation, Fecal Azoreductase Activity and Fecal Mucin in the Elderly. Annals of Nutrition and Metabolism, 46,159–162.
    
58. Ouwehand, A.C., Tolkko, S., and Salminen, S., 2001. The Effect of Digestive Enzymes on the Adhesion of Probiotic Bacteria In Vitro.
    Journal of Food Science 66 (6), 856-859.
    
59. Parassol, N., Freitas, M., Thoreux, K., Dalmasso, G., Bourdet-Sicard, R., and Rampal, P., 2005. Lactobacillus casei DN-114 001 Inhibits the Increase in Paracellular Permeability of Enteropathogenic Escherichia coli-Infected T84 Cells. Research in Microbiology, 156, 256–262.
    
60. Reid, G., 1999. The Scientific basis for probiotic strains of Lactobacillus. Applied and Environmental Microbiology, 65, 3763-3766.
    
61. Reid, G., Jass, J., Sebulsky, M. T., and Mc Cormick, J. K., 2003. Potential uses of probiotics in clinical practice. Clinical Microbiology Reviews, 658-672.
    
62. Rolfe, R.D., 2000. The Role of Probiotic Cultures in the Control of Gastrointestinal Health. The Journal of Nutrition, 130, 396S–402S.
    
63. Ross, R.P., Desmond, C., Fitzgerald, G.F., and Stanton, C., 2005. Overcoming the technological hurdles in the development of probiotic foods. Journal of Applied Microbiology, 98, 1410–1417.
    
64. Roy, D., 2001. Media for Isolation and Enumeration of Bifidobacteria in Dairy Products. International Journal of Food Microbiology, 69, 167-182.
    
65. Roy, D., 2001. Media for the isolation and enumeration of bifidobacteria in dairy products. International Journal of Food Microbiology, 69,167–182.
    
66. Saarela, M., Virkajarvia,I., Alakomia,H.L., Sigvart-Mattilab, P., and Matto. J., 2006. Stability and functionality of freeze-dried probiotic Bifidobacterium cells during storage in juice and milk. International Dairy Journal, 16, 1477–1482.
    
67. Salminen, S., and Isolauri, E., 2006. Intestinal Colonization, Microbiota and Probiotics. Journal of Pediatrics, 149, S115-S120.
    
68. Saxelin, M., Tynkkynen, S., Mattila-Sandholm, T., and De Vos, W.M., 2005. Probiotic and Other Functional Microbes: From Markets to Mechanisms. Current Opinion in Biotechnology, 16, 204–211.
    
69. Schrezenmeir, J., and De Vres, M., 2001. Probiotics, Prebiotics and Symbiotic-Approaching a Definition. American Journal of Clinical Nutrition, 73, 361S–364S.
    
70. Servin, A.L., 2003. Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Practice & Research Clinical Gastroenterology,17 (5), 741–754.
    
71. Shuhaimi, M., Yazid, A.M., Ali, A.M., Ghazali, M.H., Zaitun, H., and Nur Atiqah, N.A., 1999. Acid adaptation of bifidobacteria isolated from infants stools to simulated pH of human stomach. Pak, J. Biol. Sci., 2(4), 1203-1206.
    
72. Stadler, M., and Viernstein, H., 2003. Optimization of a formulation containing viable lactic acid bacteria. International Journal of Pharmaceutics, 256, 117–122.
    
73. Sullivan, A., and Nord, C.E., 2002. The Place of Probiotics in Human Intestinal Infections. International Journal of Antimicrobial Agents, 20, 313-319.
    
74. Surawicz, C.M., 2003. Probiotics, antibiotic-associated diarrhoea and Clostridium difficile diarrhoea in humans. Best Practice and Research Clinical Gastroenterology, 17, 775-83.
    
75. Szajewska, H., Ruszczynski, R., and Radzikowski, A., 2006. Probiotics in the Prevention of Antibiotic-Associated Diarrhea in Children: A Meta-Analysis of Randomized Controlled Trials. Journals of Pediatrics, 149, 367-372.
    
76. Takahashi, N., Xiao, J.Z., Miyaji, K., Yaeshiima, T., Hiramatsu, A., Iwatsuki, K., Kokubo, S., and Hosono, A., 2004. Selection of Acid Tolerant Bifidobacteria and Evidence for a Low-pH-Inducible Acid Tolerance Response in Bifidobacterium longum. Journal Dairy of Research, 71, 340–345.
    
77. Tannock, G.W., 2004. A Special Fondness for Lactobacilli. Applied and Environmental Microbiology, 70, 3189–3194.
    
78. Taumola, E., Playne, M., Salminen, S., 2001. Quality Assurance Criteria for Probiotic Bacteria. American Journal of Clinical Nutrition.73, 393S-398S.
    
79. Teraguchi., Uehara, S.M., Ogasa, K., and Mitsuoka., 1978. Enumeration of Bifidobacteria in Dairy Products. Japanese Journal of Bacteriology, 33(6), 753.
    
80. Tharmaraj, N., and Shah, N.P., 2003. Selective Enumeration of Lactobacillus delbrueckii ssp. bulgaricus,
    Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria. Journal of Dairy Science, 86, 2288–2296.
    
81. Todd, R.K., 2000, Probiotic Bacteria: Today and Tomorrow. The Journal of Nutrition, 130, 415S–416S.
    
82. Tuomola, E.M., and Salminen, S.J., 1998. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. International Journal of Food Microbiology, 41, 45–51.
    
83. Van de Casteele, S., Vanheuverzwijn, T., Ruyssen, T., Van Assche, P., Swings, J., and Huys, G., 2006. Evaluation of culture media for selective enumeration of probiotic strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese starters. International Dairy Journal, 16, 1470–1476.
    
84. Van Niel, C.W., Feudtner, C., Garrison, M.M., and Christakis, D.A., 2002. Lactobacillus Therapy for Acute Infectious Diarrhea in Children: a Meta-Analysis. Pediatrics, 109, 678–684.
    
85. Vanderhoof, J.A., Whitney, D.B., Antonson, D.L., Hanner, T.L., Lupo, J.V., Young, R.J., 1999. Lactobacillus GG in the Prevention of Antibiotic-Associated Diarrhea in Children. Journal of Pediatrics, 135, 564-568.
    
86. Vernazza, C.L., Bodun, A.R., and Gibson, G.R., 2006, Prebiotics: Development and Application. John Wiley and Sons Ltd, 1-28.
    
87. Vernazza, C.L., Gibson, G.R., and Rastall, R.A., 2006. Carbohydrate Preference, Acid Tolerance and Bile Tolerance in Five Strains of Bifidobacterium. Journal of Applied Microbiology, 100, 846–853.
    
88. Vinderola, C.G., and Reinheimer, J.A., 1999. Culture Media for the Enumeration of Bifidobacterium bifidum and Lactobacillus acidophilus in the Presence of Yoghurt Bacteria. International Dairy Journal, 9(8), 497-505.
    
89. Vinderola, C.G., and Reinheimer, J.A., 2000. Enumeration of Lactobacillus casei in the Presence of L. acidophilus, Bifidobacteria and Lactic Starter Bacteria in Fermented Dairy Products. International Dairy Journal, 10, 271-275.
    
90. Waterman, S.R., and Small, P.L., 1998. Acid-sensitive enteric pathogens are protected from killing under extremely acidic conditions of pH 2.5 when they are inoculated onto certain solid food sources. Appl Environ Microbiol, 64, 3882–3886.
    

My Thesis: RESULTS AND DISCUSSION

CHAPTER 4

RESULTS AND DISCUSSION

 

4.1 Colony Morphology on MRS, MRS-bile and MRS-LP agar

 

MRS agar was used to enumerate total lactic acid bacteria in product A, B and C while MRS-LP and MRS-bile were used to enumerate L. acidophilus, L. casei and bifidobacteria differentially in the multiple strains product, B and C. The differential enumeration was based on the colony morphology on the media. Differential enumeration of L. acidophilus, L. casei and bifidobacteria was not required for product A because it contains only one strain of L. acidophilus. Table 4.1 shows the colony morphology on MRS, MRS-bile and MRS-LP agar.

 

Table 4.1: Colony morphology on MRS, MRS-bile and MRS-LP agar.

Media	Microorganism1	Colony
		Diameter (mm)	Color
MRS agar	total lactic acid bacteria	0.9 – 3.0	white
MRS-bile	L. acidophilus L. casei	0.9 - 1.5 0.9 - 1.3	light brown white creamy
MRS-LP	L. casei bifidobacteria	varies	varies

¹ target microorganism of the differential media

 

MRS agar was incubated in aerobic and anaerobic condition in order to obtained total aerobes and total anaerobes of lactic acid bacteria, respectively. Lactic acid bacteria in MRS agar yielded white round colonies ranging in diameter from 0.9 to 3 mm (figure 4.1). The colony appearance aerobes and anaerobes was similar for product A, B and C. In this study, product B and C, the total aerobes and total anaerobes represent the cells count of lactic acid bacteria whereas total anaerobes represent the cells count of L. acidophilus in product A.

 

Figure 4.1: Colony appearance of lactic acid bacteria in MRS agar for product B.

 

 

 

 

 

 

 

 

MRS-bile was used for differential enumeration of L. acidophilus and L. casei (Vinderola et al., 1999; Vinderola et al., 2000). Figure 4.2 shows the colony appearance of L. acidophilus and L. casei on MRS-bile agar. L. acidophilus yielded light brown colonies, ranging in diameter from 0.9 to 1.5 mm and L. casei yielded round white creamy colonies, ranging in diameter from 0.9 to 1.3 mm. Hence, both microorganisms yielded different color of colonies. The colony morphology of both microorganisms obtained from the MRS-bile agar was in accordance with Vinderola et al. (2000). He confirmed that these two distinct types of colonies were L. acidophilus and L. casei, respectively, as the colonies were examined on cell morphology (phase contrast, 1000x) and catalase test. Therefore, he reported that the differential cells count of L. acidophilus and L. casei can be assessed by their distinguishable colonies in the agar. As a result, in this study, MRS-bile agar provides a reliable bacterial count of L. acidophilus and L. casei.

 

Figure 4.2: Colony appearance of L. acidophilus and L. casei in MRS-bile agar for product B.

 

 

 

 

 

 

 

 

MRS-LP was used for differential enumeration of bifidobacteria and L. casei, based on their distinguishable colonies (Vinderola et al., 1999; Vinderola et al., 1999). In this study, product B and C yielded various sizes of colonies on MRS-LP (figure 4.3). The colonies did not give two distinct types of colonies and the identity of the colonies was obscured. The colonies were Gram positive and appeared as rod shape or Y-shape under light microscope (x1000). Although some of the colonies appeared as Y-shape (distinctive morphology of bifidobacteria) under light microscope, this did not verify the identity of these colonies because biochemical tests are required for verification. Concurrently, the various sizes of colonies yielded did not conform to Vinderola et al. (1999) and MRS-LP failed to enumerate bifidobacteria and L. casei. In this study, the differential cells count of bifidobacteria was not obtained.

 

The differential agar strongly relies on the differences in colonial morphology, which is not always a stable phenotypic trait to identify and quantify probiotic organisms in a product. The failure of MRS-LP agar as differential agar could be also due to the presence of others microorganism instead of the target microorganisms, L. casei and bifidobacteria. Product B and C consisted of S. thermophilus, L. acidophilus, L. bulgaricus and L .rhamnosus. Some of them might not be inhibited by the lithium propionate in the differential agar and thus they were present collectively with the target microorganisms in the MRS-LP. In this regard, growth requirements may vary strongly between taxa and this appears that the selective enumeration of all industrially important bifidobacteria by the use of one selective medium is very difficult to achieve (Casteele et al., 2006). As a consequence, the medium choice had to be considered on different aspects as it is very unlikely that the proposed media are suitable for the desired application.

 

Figure 4.3: Colony appearance of microorganism in MRS-LP agar for product B.

 

 

 

 

 

 

 

 

4.2 Viability of pharmaceutical probiotic products

 

In this study, the viability of pharmaceutical probiotic products was assayed by plate count method. Table 4.2 shows the viability of pharmaceutical probiotic products obtained from MRS (aerobic and anaerobic) and MRS-bile agar.

 

Table 4.2: Enumeration of total aerobes, total anaerobes, L. acidophilus and L. casei obtained from MRS agar and MRS-bile at 37°C after 72 hours.

Brand	Viable cell count (log10 CFU/g)1
	MRS		MRS-bile
	Total aerobes	Total anaerobes	L. acidophilus	L. casei
A	ND2	7.37±0.00	ND	ND
B	9.75±0.04	9.82±0.01	9.52±0.09	8.92±0.03
C	0	4.79±0.14	0	0

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates).

² ND: Not determined

 

In this study, all the products (A, B and C) did not meet the claimed viability which stated on the label. Nevertheless, product A and B contained more than 10⁶ CFU/g (minimum requirement for probiotic product) whereas product C contained less than 10⁶ CFU/g.

 

Furthermore, the viability of single strain product (A) of L. acidophilus was assessed as total anaerobes because L. acidophilus grows well in the anaerobic condition (Lin et al., 2006). The viability of multiple strains probiotics products (B and C) was assessed as total aerobes and total anaerobes. The lactic acid bacteria in the products were grow in both aerobic and anaerobic condition because some of them were microaerophilic and strictly anaerobic. However, in product C, only anaerobic incubation yielded growth which meant that the probiotics were strictly anaerobic.

 

Moreover, the viability loss of the probiotic products may be caused by temperature fluctuation during stock delivery, storage and upon purchasing. Correct storage temperature of probiotic preparations is essential to maintain viable populations of freeze-dried probiotic bacteria. Besides, the rate of freeze drying and cryoprotectant also affect the viability of the products since product A, B and C are in freeze dried form. During freeze-drying, osmotic shock and membrane injury may happened. Large ice crystal formation caused damage to the cell membrane and hence reduced its viability. Besides, absence or inefficacy of cyroprotectant may lead to low viability in the products (Heckly, 1985; Egawa et al., 2005; Saarela et al., 2006; Morgan et al., 2006).

 

In addition, the prices of the pharmaceutical probiotic products do not correspond to the viability of the product. This is because product C is the most expensive but contained the lowest viability. In contrast, product A was the cheapest but rank second highest in viability whereas product B was the secondly expensive and contained the highest viability.

Although all the products did not meet the label claim for viable cells count, two of the products contained more than 10⁶ CFU/g (minimum requirement for probiotic product). Therefore, product A and B retain the criterion of 10⁶ CFU/g and may confer health effects upon ingestion.

 

Eventually, the prophylactic and therapeutic effects of probiotics will be effective only if the probiotics remain as high number of viable cells in the gastrointestinal tract after consumption. The minimum viable cells that is required is 10⁶ colony forming unit per gram (CFU/g) (Dave et al., 1997; Kailasapathy et al., 2000). Product A and B contained more than 10⁶ CFU/g and hence, these probiotics may exert its effect if they were able to tolerate to low pH. Subsequently, this gave the rationale of acid tolerance test.

4.3 Direct Count Method: Dye Exclusion Test

 

The dye exclusion test was tested in order to obtain direct viable counts of bacteria in the products. From the result, clumping of cells were observed under the microscope (x 400) and hence, the cells count could not be determined. Although the sample was centrifuged prior the test, the similar results were obtained. Thus, this method did not provide a reliable way of direct viable cells count.

 

The factors that affect the efficacy of this method could be caused by the presence of adjuncts (cryoprotectants) in the pharmaceutical probiotics which causes the cells clumped together. Moreover, the adjuncts may be too dense to be separated through centrifugation. Even after the sample centrifuged, the adjunct could be retained in the bacteria pellet. Since this method gave a negative result and was not reliable, this method was dropped from the study.

Therefore, the viable cells count of the probiotics was assessed by plate count method. The CFU per gram provide the viable cells count of probiotics throughout this study.

4.4 Acid Tolerance of Pharmaceutical Probiotics

 

In order to evaluate the acid tolerance of pharmaceutical probiotics, product A, B and C were tested in acidified-PBS in pH 2, 3, 4 and 6. Acidified-PBS was used instead of acidified-MRS broth because MRS broth may provide protection to bacteria by providing energy and metabolic precursors, thus potentially enhancing bacterial survival (Corcoran et al., 2005). Products A, B and C were tested for 0, 1.5 and 3 hours to simulate pH of stomach by taking into account the general gastric transit time. Table 4.3 shows the viable cells count of product A for acid tolerance test. From the study, there was significant decreased in viability at pH 2 and 3 while no significant decreased in viability at pH 4 and 6 in product A. The tolerance to pH 4 and pH 6 for probiotics product A were better than pH 2 and pH 3. At pH 2, viability of product A decreased approximately 3-fold from 6.77 log₁₀ CFU/g to 6.28 log₁₀ CFU/g after 3 hours exposure. At pH 3, viability of product A decreased approximately 2-fold from 7.07 log₁₀ CFU/g to 6.80 log₁₀ CFU/g after 1.5 hours exposure and the viability did not further decrease for another 1.5 hours. Although there were decreased in viability at pH 2, the viable cells count still maintained above 10⁶ CFU/g (minimum requirement for probiotic products) after 3 hours exposure. Thus, product A which contains probiotic strain L. acidophilus was tolerant to the simulated gastric pH.

 

Table 4.3: Enumeration of L. acidophilus in product A following exposure to pH 2, 3, 4 and 6 after 0, 1.5 and 3 hours incubation. The values are the mean of three determinations.
¹

pH	Viable cells count (log10CFU/g)2
	0hr	1.5hr	3.0hr
2	6.77±0.06a	6.99±0.07b	6.28±0.02c
3	7.07±0.16ab	6.80±0.12 abc	6.76±0.02bc
4	7.08±0.07abc	7.21±0.11abc	7.17±0.17abc
6	7.38±0.13abc	7.37±0.12abc	7.32±0.06abc

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates/products). Means in row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar.

 

The method for acid tolerance test was similar for all the products except in product B and C, the enumeration of probiotics included differential counts of L. acidophilus and L. casei. Table 4.4 shows the viable counts in product B following exposure to pH 2 for 0, 1.5 and 3 hours. In product B, there was significant decreased in total viability at pH 2 after 3 hours. The viable cells count of total aerobes decreased approximately 160-fold from 9.00 log₁₀ CFU/g to 6.80 log₁₀ CFU/g after 3 hours exposure. The viable cells count of total anaerobes decreased 130-fold from 9.47 log₁₀ CFU/g to 7.37 log₁₀ CFU/g after 1.5 hours exposure and the viability did not further decrease for another 1.5 hours. The viable cells count of L. acidophilus in product B decreased approximately 180-fold from 9.13 log₁₀ CFU/g to 6.88 log₁₀ CFU/g after 3 hours exposure. The viable cells count of L. casei decreased 120-fold from 8.90 log₁₀ CFU/g to 6.81 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. Although there were decreased in total viability at pH 2, the viability still maintained above 10⁶ CFU/g (minimum requirement for probiotic products) after 3 hours exposure. Thus, pharmaceutical probiotics of product B was tolerant to the simulated gastric pH.

 

Table 4.4: Enumeration of viable count in product B following exposure to pH 2 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g) 1
	0hr	1.5hr	3.0hr
total aerobes2	9.00±0.11a	6.40±0.07bc	6.80±0.34bc
total anaerobes2	9.47±0.04a	7.37±0.03bc	7.34±0.05bc
L. acidophilus3	9.13±0.09a	6.58±0.36bc	6.88±0.16bc
L. casei3	8.90±0.23a	6.81±0.22bc	6.81±0.22bc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.5 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 3 for 0, 1.5 and 3 hours incubation. There was significant decreased in viability at pH 3 for 0, 1.5 and 3 hours for total aerobes, total anaerobes and L. casei. The viable counts of total aerobes decreased 3-fold from 9.50 log₁₀ CFU/g to 9.08 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. The viable counts of total anaerobes decreased 2-fold from 9.44 log₁₀ CFU/g to 9.18 log₁₀ CFU/g after 3 hours exposure. Likewise, the viable cells count of L. casei decreased 3-fold from 9.22 log₁₀ CFU/g to 8.80 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. In contrast, L. acidophilus showed no significant decrease at pH 3 and the viability was well maintained for 3 hours exposure.

Table 4.5: Enumeration of viable count in product B following exposure to pH 3 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
total aerobes2	9.50±0.08a	9.08±0.17bc	9.01±0.07bc
total anaerobes2	9.44±0.02a	9.27±0.12abc	9.18±0.04bc
L. acidophilus3	8.76±0.39abc	8.86±0.09abc	8.86±0.09abc
L. casei3	9.22±0.09a	8.80±0.04bc	8.80±0.04bc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.6 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 4 for 0, 1.5 and 3 hours incubation. In product B, there was significant decreased in viability at pH 4 for 0, 1.5 and 3 hours for total anaerobes only whereas total aerobes, L. acidophilus and L. casei showed no significant decrease. The viable cells count of total anaerobes decreased approximately 1.5-fold from 9.43 log₁₀ CFU/g to 9.27 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. From the result, the viable cells count of aerobes, L. acidophilus and L. casei were well maintained at pH 4 for 3 hours exposure. Moreover, pH 4 did not cause detrimental effect on L. acidophilus and L. casei of product B.

 

Table 4.6: Enumeration of viable count in product B following exposure to pH 4 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
total aerobes2	9.67±0.17ab	9.46±0.02abc	9.24±0.02ac
total anaerobes2	9.43±0.05a	9.27±0.08bc	9.16±0.03bc
L. acidophilus3	9.42±0.02abc	9.00±0.32abc	8.87±0.50abc
L. casei3	8.62±0.15abc	8.30±0.30abc	8.70±0.00abc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.7 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 6 for 0, 1.5 and 3 hours incubation. The probiotics of product B showed no significant decreased in viability to pH 6 for 3 hours exposure. The viable cells count of aerobes, anaerobes, L. acidophilus and L. casei were well maintained at pH 6 for 3 hours exposure. Moreover, pH 6 did not cause detrimental effect on pharmaceutical probiotics of product B.

 

Consequently, both pH 4 and pH 6 did not cause detrimental effect on aerobes, L. acidophilus and L. casei of product B after 3 hours of acidified-PBS exposure. This result was in agreement with Liong et al. (2005) where they concluded that L. acidophilus and L. casei strains were able to survive under acidic environment. Nevertheless, total anaerobes of product B were maintained only at pH 6. Although the viability of product B was decreased to variable extent, its viability was still maintained above 10⁶ CFU/g (minimum requirement for probiotic products). Therefore, pharmaceutical probiotics of product B survived the pH 2, 3, 4 and 6.

 

Table 4.7: Enumeration of viable count in product B following exposure to pH 6 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
Total aerobes2	9.75±0.04abc	9.64±0.05abc	9.55±0.19abc
Total anaerobes2	9.82±0.01abc	9.66±0.06abc	9.57±0.21abc
L. acidophilus3	9.85±0.02abc	9.77±0.04abc	9.81±0.06abc
L. casei3	9.33±0.54abc	9.66±0.47abc	9.65±0.50abc

 

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

 

On the other hand, product C was tested for acid tolerance although the initially viability was lower than 10⁶ CFU/g. Table 4.8 shows the viable cells count in product C for acid tolerance test. The acid tolerance test showed that the pharmaceutical probiotics of product C did not survive the pH 2, 3 and 4. However, in pH 6 the viability was maintained and shown no significant decreased after 3 hours exposure. From the results, probiotics of products C did not tolerate to low pH.

Table 4.8: Enumeration of viable cells count in product C following exposure to pH 2, 3, 4 and 6 after 0, 1.5 and 3 hours incubation. The values are the mean of three determinations.
¹

pH	Viable cells count (log10CFU/g)2
	0hr	1.5hr	3.0hr
2	0	0	0
3	0	0	0
4	0	0	0
6	4.79±0.01abc	4.71±0.01abc	4.69±0.01abc

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates/products). Means in row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar under anaerobic condition.

 

 

 

 

 

 

 

 

 

 

 

 

 

This study compared the effects of different pH on the viability of pharmaceutical probiotics in three different brands during 3 hours of acidified-PBS exposure simulating the gastric pH. In this regard, probiotics of product C did not survive at pH 2 (the lowest pH in this study) compared to product A and B. As shown in graph 4.1, product B had the highest tolerance to pH 2 compare to product A. From the result, the varied degree of acid tolerance among the probiotic products could be influenced by the different strains incorporated in the particular products (Cotter et al., 2003). Product B could be consisted of several good strains of probiotics that exhibit better survivability to low pH whereas in product C, the probiotic strains were poor and thus did not survived in low pH. Moreover, the acid tolerance of product B was due to the overall effects exhibited by all the microorganisms present inside. Hence, acid tolerance of product A was weaker than product B because its survivability in low pH was exhibited by the single strain of L .acidophilus.

 

In addition, the prices of the products do not correspond to the acid tolerance of its probiotics. Since product C is the most expensive but contained the lowest acid tolerance. In contrast, product B was the secondly expensive and contained the highest acid tolerance whereas product A was the cheapest but rank second highest in acid tolerance.

 

Low pH condition of stomach causes loss of viability and cell damage. Cell damage is envisioned as the derangement of membrane structures as well as solute leakage from the cell.
It was reported that L. casei leak magnesium only at pH less than 3. This phenomenon was comparable with the present study as the viability of L. casei for product B was affected at less than 3 (pH 2 and 3). In addition, the susceptibility to
membrane damage caused by acidification is varied between species and correlate with the degree of acid tolerance (Hutkins et al., 1993).

 

There are various underlying factors and mechanisms that could affect the acid tolerance of probiotics. The survival of probiotics is high when there are availability of nutrients, low or absence of inhibiting compounds and maintenance of hydrogen ion concentration above the level that a specific strain can tolerate. Low pH is growth limiting for probiotics and loss of cell viability may also occur in cells that are held at low pH. The acid tolerance of lactobacilli or probiotic is attributed to the presence of a constant gradient between extracellular and cytoplasmic pH (or intracellular pH). When the internal pH reaches a threshold value, cellular functions are inhibited and the cells die (Corcoran et al., 2005). Moreover, the optimum growth of bacteria occurs within a specific pH range, depending upon the species of bacterium (Nannen et al., 1991). This explains the reason why the pharmaceutical probiotics in this study have varying degree of acid tolerance.

 

In general, bacteria are equipped with a number of mechanisms that confer acid tolerance. These include proton translocation, arginine deaminase (ADI) pathway, amino acid decarboxylation-antiporter reactions, and the citrate transport system. These mechanisms will activated when bacteria are confronted with a change in the pH. Nevertheless, among these mechanisms, the one being significant to lactic acid bacteria or probiotics is the proton translocation. This is one of the important mechanism of which probiotics used for its pH homeostasis. The F₀F₁ ATPase functions to maintain a favorable intracellular pH and protect cells during exposure to acidic environment by translocating protons to the environment at the expense of ATP. F₀F₁-ATPase is induced at low pH, and regulation appears to occur at the transcriptional level (Corcoran et al., 2005). The membrane bound proton-translocating ATPase (H⁺-ATPase) extrudes protons out of the cell via ATP hydrolysis. The expulsion and movement of protons from the cytoplasm into the medium of the cells (more acidic) is against the concentration gradient and hence energy is needed. The main function of H⁺-ATPase in glycolytic, nonrespiring bacteria (lactic acid bacteria) is the maintenance of ∆pH (pH gradient) and functions as a proton pump (Hutkins et al., 1993; Matsumoto et al., 2004).

 

Furthermore, the undissociated form of an organic acid is assumed to be the most toxic form for microorganisms compare to inorganic acid, HCl. Organic acids inhibit microorganisms by entering the cell in the undissociated form and then dissociating within the cell which lead to acidification of the cytoplasm and collapse of the proton motive force, resulting in inhibition of nutrient transport. Therefore, the uses of acid in acid tolerance test simulating the stomach condition is an important consideration (Hutkins et al., 1993; Matsumoto et al., 2004; Cotter et al., 2003).

 

When probiotics are to be allowed to grow in acidic condition, it will perform the acid tolerance mechanism to maintain the cytoplasmic pH relatively constant over a wide range of environmental pH. And this will restore its pH homeostasis. But, when there is a pH shift during the stationary phase of probiotics, the expression of proteins will be modified. This inducible survival mechanism is known as acid tolerance response. Hence, it is advisable to grow cells for probiotics purposes at lower pH in order to improve the resistance of the cells to acid stress during the acidic stomach passage (Hutkins et al., 1993; Matsumoto et al., 2004; Collado et al., 2006).

 

 

 

The results from present study indicate that the survival of pharmaceutical probiotics in stomach acidity was varied among the products. The sum of these effects were influence by the strain of probiotics, the pretreatment of probiotics before undergo processing and its mechanisms of the acid tolerance.

 

Besides, there are other factors that affect the survival of probiotics in human stomach. These factors are due to various chemical components present in human stomach. These include the gastric enzyme such as pepsin as well as digested foods or nutrient. The foods matrix may protect the probiotics from the digestive enzyme as well as the nature of food will affects the transit time through the stomach. Basically, food remains in the stomach between 2 and 4 hour however, liquids empty from the stomach faster than solids, and only take about 20 minutes to pass through the stomach (Ouwehand et al., 2002; Holzapfel et al., 1998; Huang et al., 2004).

 

Although pH could be used as a suitable parameter for selection of probiotic strains, the pharmaceutical probiotics are consumed in capsule form. As such, the presence of capsule may improve the viability of microorganisms during gastric transit. This suggest that low-acid tolerant strains need not be excluded from probiotic application, providing they can be delivered to the intestine in high numbers, and preferably as part of a buffered food or encapsulated delivery system (Huang et al., 2001).

4.5 Morphology Identification

    

The colonies yielded in MRS and MRS-bile agar was tested for Gram staining. From the results, all the microorganisms from MRS agar were Gram positive. Rod and cocci shape were found in product C because product C was claimed to contained lactobacilli and S. thermophilus (table 3.1) In contrast, rod shape was found in product A and B because both of them were claimed to contained lactobacilli (table 3.1). Furthermore, all the microorganisms from MRS-bile agar were Gram positive rod shape as this media was used to enumerate L. acidophilus and L. casei differentially.