Keywords

Probiotic;Health claims; Food; Starter cultures.

Introduction

Fermentation employing lactic acid bacteria is the oldest,simple and safest means of food preservation [1] and relatively recently their efficacy to exhibit health benefits have been explored [2]. With the realization of the link between diet and health, a worldwide consumer’s inclination towards functional foods that possess certain health properties besides basic nutrition have been noted. An intense acceptance of functional foods due to consumer’s demand, social attitudes, scientific evidence of the human health benefits of a particular ingredient and commercially driven interest to add value to existing foods were observed [3].

Probiotics may be defined as “live microbial food ingredient that, when ingested in sufficient quantities, exert health benefits on the consumer” [4]. Probiotics are now emerged as an important category of food supplement and could be found in conventional, dietary supplements and medicinal foods [5] in many countries including Japan, Europe and USA [6]. Significance of human gut microbiota in health restoration and maintenance have led acceptance of probiotics as functional foods [2] in current era of self-care and complementary medicine [7, 8] and comprises approximately 65% of the world’s functional food market [9]. Owing to diverse prophylactic properties, probiotic foods could fall into the category of functional foods [3] and consumption of functional probiotic bacteria is increasing due to promotion of gut health, disease prevention and therapy [10]. Beneficial health effects extend by probiotics is due to maintenance of the equilibrium of indigenous microbiota [11] with the growth inhibition of pathogenic microorganisms and boosting of innate and acquired immunity [12]. It has been established that viability and metabolic activities of probiotics during food processing, at the point of sale [13, 14] and in host gastro-intestinal tract [15] are essential for extending health benefits. For long-term existence of probiotic as functional foods in the world market, it becomes imperative to ensure their higher viability till consumption and ability to exhibit probiotic effect [3]. Further well-designed placebo-controlled studies are emerging for determining the optimal dose, duration of treatment, selection probiotic strains, their mode of actions [16] and efficacy of multi-strain preparations [17] prior to their recommendations for therapeutic or preventive use. In the present endeavor, an attempt has been made to highlight the microbiological considerations for probiotic selection to ensure its safe application in probiotic supplemented foods capable of exhibiting prophylactic characteristics.

Viability of Probiotic Cultures

Significance of Probiotic Viability

For exhibiting prophylactic properties, cultured milk products must retain sufficient population of viable organisms throughout its anticipated shelf-life. Ingestion of acidophilus milk containing 3x107cells/ml L. acidophilus for 30 day induced a fall in blood serum cholesterol level in human volunteers [18]. Ingestion of yoghurt containing 108 cfu/g B. longum [19] and hydrolyzed whey formulae containing 1x109 cfu/g B. lactis [20] induced significant reduction in total serum cholesterol in humans and modify infant’s gut microbiota, thereby alleviating allergic inflammation.

An elevation in bifidobacteria counts as well as a decline in enterobacteria in mice cherishing bifidus milk containing 107 cells of B. longum for 14 days was noted [21]. Animal and infant feeding trials revealed a decline in coliforms and an increment in faecal bifidobacteria and/or lactobacilli due to ingestion of bifidus milk [22], Propiono-Acido-Bifido [PAB] milk [23, 24] and dietetic yoghurt [25] containing B. bifidum [108 cfu/ml]. Ingestion of fermented milk containing 5x107 cfu/ml L. acidophilus and L. casei by human volunteers [26] or bifidus milk containing 108 cfu/g B. bifidum by infants [22] induced an elevation in faecal lactobacilli [7.59-8.93 log cfu/ml] and bifidobacteria (2.2x108-19.8x108cfu/g), respectively.

Lactobacilli supplemented in milk at a level of 109 viable/day [27] or 1011 cfu/day [28, 29] were efficacious in reducing the faecal β-glucuronidase and β-glucosidase activity in human subjects responsible for carcinogenesis. Decline in nitroreductase activity during ingestion and its retention at a low level after cessation of intake of fermented milk product containing L. acidophilus [107 cfu/g], B. bifidum ([108 cfu/g) and Lactococcus lactis (108 cfu/g) and Lactococcus lactis subsp. cremoris (108 cfu/g) were noted [30].

An improvement in lactose digestion due to ingestion of milk supplemented with L. acidophilus (2.5x106 to 2.5x10,8cfu/ml) was observed [31]. Human trials revealed lowest breath hydrogen (9.9, 22.8, 50.2 ppm) due to ingestion of cultured yoghurt in contrast to heated cultured yoghurt or direct acid yoghurt, respectively [32]. Decrement in viable population (3x108/g to 3.4x106/g) and lactase activity (0.64 to 0.07 units/g) due to pasteurization [33] and better tolerance of non-pasteurized yoghurt than pasteurized yoghurt by lactase-non-persistent individuals [34, 35] indicate sigS. Sarkar, International Journal of Microbiology & Advanced Immunology 2013, 1:102 3 nificance of viable population.

It has been established that to achieve health benefits through ingesting cultured milk products especially yoghurt [36, 37], probiotic cultures must retain its viability at a level of >106 cfu/g [38]. It has been mentioned that the viability of microorganisms must be retained both at the end of incubation as well as at the date of expiry of the product [39]. Suggested daily intake being >108/g [40, 41], probiotic products must be consumed regularly in sufficient quantities to deliver the relevant dosages of live bacteria to the gut [42]. The recommended intake is 300-400g/ week [43] or 100g/day [44]. Recommended viable population of probiotic to be present in food by different agencies is depicted in Table I.

Viability of Probiotics in Probiotic Foods

Several reports indicated poor viability of probiotics in health products [40, 49] and often present at levels lower than those claimed on label [50, 51]. Survey reports on fermented functional foods and health-care products indicated lower microbial contents than the labelled claim in few health-care products, whereas for bio-yoghurts no indication microbial content was furnished [52].

Bifidobacterium sp. could not be detected in drinking yoghurt-containing probiotics and reported that the identified strains do not always correspond to those declared on the label [53]. Bifidobacteria could be detected at a level 0.0 to 6.0 log cfu/ml only in 76% of the analyzed samples of bio-yoghurt containing Bifidobacteria sp. [54, 55] and respectively in 90 and 50% of samples during purchase and date of expiry [56]. Recent market survey in Columbia on bio-yoghurts revealed that though the products had a total viable cell population of 107cfu/ml, however Bifidobacterium could be recovered only in 14.29 % samples [57].

Presence of viable population of L. acidophilus and Bifidobacterium sp. at a level lower than the recommended level (106cfu/g), by the expiry date of most of the market probiotic yoghurts have been mentioned [58, 59]. Poor viability and large deviation in viability of bifidobacteria in yogurt have been mentioned [60] and are present at non-detectable levels or at a level of 104 -107 cfu/ml [58] or 106 cfu/ml [61]. Bifidobacteria were less acid tolerant than L. acidophilus [62] and were detected at a level of 10⁶ cfu/ml, respectively in 14 and 24% of yoghurt samples [61] and both retained their viability at a level of >105cfu/ml during storage [63]. Lower bifidobacterial population (< 103 cfu/g)than L. acidophilus (< 103-108 cfu/g) were detected in few Australian yoghurt containing probiotic cultures [64]. However, another investigation indicated B. bifidum was to be more resistant to yoghurt environment than L. acidophilus [65, 66] and the counts declined from 1.54±0.45x109 to 0.38±0.02x109 cfu/ml during 15 days storage [67]. Stability of bifidobacteria and L. acidophilus in yoghurt environment is pH dependent. Decline in viability of bifidobacteria and L. acidophilus were negligible at pH 5.0 but population declined by 0.1-7.6 log cycles and 1.6-6.2 log cycles, respectively at pH 4.0 [68]. Micro-aerophilic and anaerobic characteristics of L. acidophilus and Bifidobacteria sp. render them susceptible to oxygen contained in the yoghurt, resulting in their poor viability during its anticipated shelf-life [69]. It has been annunciated that the initial concentration of yoghurt cultures must be maintained at 108-109 cells/ml in milk for sustaining therapeutic dosage up to 21 days/ 5 °C [70] due to loss of viability by heat, pressure, low water activity and high acidity [71].

---

×

Table1:

Close

Table 1: Recommended probiotic viability in probiotic foods

---

Besides instability of probiotic cultures in product itself, viability is also lost during its transit through intestinal tract. Viability of lactic acid bacteria is reported to get influenced by gastric pH, digestive enzymes, bile salts [72] and must be adapted to the intestinal environment for its prolonged survival [73], as only 20-40% probiotic cultures survive the gastric transit [74]. Though appreciable growth of B. bifidum and L. acidophilus in presence of bile salts [75] and better stability of former organism than Lactobacillus delbruekii subsp bulgaricus in the intestinal environment have been denoted but their survivality declined during passage through intestinal tract [25]. It has been annunciated that ingestion of fermented milk containing probiotic cultures resulted in survival of 23.5±10.4% bifidobacteria [76], 30% B. bifidum, 10% L. acidophilus [77], 6.54-9.8% bifidobacteria and 4.4-7.45% lactobacilli [24, 25] in the ceacum.

It is therefore necessary to ensure retention of viability of probiotic organisms both during processing, storage as well as transit through gastrointestinal tract with the objective of achieving prophylactic effects.

Factors Affecting Viability of Probiotics
Following factors affect the viability of probiotics in yoghurt during manufacture, storage and gastrointestinal tract transit.

• acid and hydrogen peroxide production by yoghurt cultures
• dissolved oxygen content of the product
• oxygen permeability through the package [78]
• concentration of lactic and acetic acids in the product [79].
• fat content of milk [66]
• heat-treatment of milk
• incubation temperature [80]
• concentration of buffers such as whey protein concentrate [81]
• physiological status of probiotic cultures added
• physical condition of product storage
• possible interactions of the product with starter cultures[82].

Microbiological Considerations for Probiotic Supplemented Foods,

Microbiological Considerations for Probiotic Selection<

Selection of probiotic cultures intended for supplementation in foods should be based upon following criteria.

• must retain the functional health characteristics for which they were originally selected [83]
• beneficial effect on the host organism
• should adhere to the mucosal epithelial cells
• should exhibit enhancement and protection of the intestinal ecology [84]
• does not have the ability to invade the host intestinal tissues and cause any infection
• sensitive to broad spectrum and commonly used antibiotics[85]
• should be isolated from the same species as its intended host
• should be able to survive transit through the gastrointestinal tract [86]
• every strain must exhibit efficacy of health benefits [87]
• must be non-pathogenic, non-toxic, and free of significant adverse side effects
• must retain stability during the intended shelf life of the product
• must contain an adequate number of viable cells to confer the health benefit
• must be compatible with product format to maintain desired sensory properties
• must be labeled in a truthful and informative manner to the consumer [88]

Microbiological Considerations for Health Claims

A ‘health claim’ is defined as “a statement, which characterizes the relationship of any substance to a disease or health-related condition, and these should be based upon well-established, generally accepted knowledge from evidence in the scientific literature and/or recommendations from national or international public health bodies [89]. Probiotic can be commercialized either as nutritional supplement, pharmaceutical or foods but the marketing as a pharmaceutical product requires significant time, complex and costly research and demonstration of well-defined therapeutic targets [90]. Obstacles in providing probiotic therapy include selection of appropriate strains, poorly regulated probiotic quality, human biological factors which impair probiotic viability, difficulties in maintaining new bacterial population in the gut and local product [91]. Various clinically relevant steps required for the acceptance of probiotics by the medical community are enumerated underneath [92].

• implementation of Guidelines for the use of probiotics
• phase I and II clinical trial data on strains and end products to prove health benefits
• use of Good Manufacturing Practices and production of high quality products
• studies which identify mechanisms of action of probiotic strains in vivo
• appropriate information dissemination about products to physicians, health professionals and lay people
• development of probiotic organisms that carry vaccines or other beneficial substances to the host
• development of anti-viral probiotics
• expansion of proven strains to benefit the oral cavity,nasopharynx, respiratory tract, stomach, vagina, bladder,and skin as well as for cancer, allergies and recoveryfrom surgery and injury

Microbiological Considerations for Safety Aspects

The usual approach for safety assessment for marketing probiotic bacteria in the United States is presumption of safety, reasoned by a long history of safe use in fermented dairy products [93]. GRAS [Generally Recognized as Safe] substances are food substances judged by qualified subject experts as safe under the intended conditions of use. It should not be assumethat all probiotics are GRAS, even if they are composed of species of Lactobacillus or Bifidobacterium [88]. In recognition of the importance of assuring safety, even among a group of bacteria that is GRAS, assessment of safety of a probiotic should be based upon the following documents.

• determination of antibiotic resistance patterns
• assessment of certain metabolic activities (e.g., Dlactate production, bile salt deconjugation)
• assessment of side-effects during human studies
• epidemiological surveillance of adverse incidents in consumers (post-market)
• strain must be tested for toxin production if the strain under evaluation belongs to a species that is a known mammalian toxin producer
• determination of hemolytic activity of strain is required if the strain under evaluation belongs to a species with known hemolytic potential [94]
• efficacy of the novel strains and the safety status of the traditional product in which they will be incorporated must be evaluated prior to their incorporation [95]
• if applicable, establishing a history of safe use based on the intended use of the species in question
• conducting toxicity or pathogenicity assessments in validated laboratory or animal models that are relevant to the species being considered, as needed [88]

Conclusion

Recently, worldwide consumer’s interest in probiotics as a functional food has increased dramatically owing to its potential human health benefits. Viability of probiotics at a desired level at the end of shelf-life of the product is the key factor for exhibiting health beneficial effects; however recent market surveys indicate their poor viability. Microbiological considerations must be given for probiotic selection to ensure its safe application in probiotic supplemented foods capable of exhibiting prophylactic characteristics. Extensive clinical trials are indicated prior to clinical application.

References

1. Molin G [2001] Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum 299v.Am J Clin Nutr 73: 380-385.
2. Kopp-Hoolihan L [2001] Prophylactic and therapeutic uses of probiotics: a review. J Am Diet Assoc 101: 229-238.
3. Stanton C, Gardiner G, Meehan H, Collins K, Fitzgerald GP,et al. [2001] Market potential for probiotic. Am J Clin Nutr 73: 476-483.
4. Donor S, Gorbach SL [2006] Probiotics: their role in the treatment and prevention of disease. Expert Rev Ant. Infec Therapy 4: 261- 275.
5. Sanders ME [2000] Considerations for use of probiotic bacteria to modulate human health. J Nutr 130: 384-390.
6. Lin DC [2003] Probiotics as functional foods. Nutr Clin Pract 18: 497-506.
7. Kaur IP, Chopra K, Saini A [2002] Probiotics: potential pharmaceutical applications. Eur J Pharm Sci 15: 1-9.
8. Sarkar S [2007] Functional foods as self-care complementary medicine. Nurt Fd Sci 37: 160-167.
9. Agarwal RA [2005] Probiotics: An emerging food supplement with health benefits. Fd Biotechnol 19: 227-246.
10. Saarela M, Lahteenmaki L, Crittenden R, Salminen S, Matilla- Sandholm T [2002] Gut bacteria and health foods - the European perspective.Int J Fd Microbiol 78: 99-117.
11. Madden JA, Plummer SF, Tang J, Garaiova I, Plummer NT,et al. [2005] Effect of probiotics on preventing disruption of the intestinalmicroflora following antibiotic therapy: a double-blind, placebocontrolled pilot study. Int Immunopharmacol 5: 1091-1097.
12. Galdeano CM, de Moreno de Leblanc A, Vinderola G, Bibas Bonet ME, Perdigon G [2007] A proposal model: Mechanisms of immunomodulation induced by probiotic bacteria. Clin Vaccine Immunol 14:485-492.
13. Kailasapathy K [2002] Microencapsulation of probiotic bacteria: technology and potential applications. Curr Iss Intest Microbiol 3:39-48.
14. Stanton C, Desmond C, Coakley M, Collins JK, Fitzgerald G, et al. [2003] Challenges facing development of probiotic-containingfunctional foods. In: Handbook of Functional Fermented Foods, Ed.Farnworth, E.R., Boca Raton, CRC Press.
15. Hamilton-Miller JM, Shah S, Smith CT [1996] Probiotic remedies are not what they seem. Br Med J 312: 55-56.
16. Goossens D, Jonkers D, Stobberingh E, van den Bogaard A,Russel, M, et al. [2003] Probiotics in gastroenterology: indications and future perspectives. Scand J Gastroenterol 239: 15-23.
17. Penner R, Fedorak RN, Madsen KL [2005] Probiotics and nutraceuticals:non-medicinal treatments of gastrointestinal diseases. CurrOpin Pharmacol 5: 596-603.
18. Khedkar CD, Garge RD, Mantri JM, Khedkar GD [1998] Studies on the hypocholesterolemic properties of acidophilus milk on human subjects of 30-40 years. J Dairying Fd Home Sci 17: 199-204.
19. Xiao JZ, Kondo S, Takahashi N, Miyaji K, Oshida K, et al. [2003] Effects of milk products fermented by Bifidobacterium longumon blood lipids in rats and healthy adult male volunteers. J Dairy Sci 86:2452-2461.
20. Kirjavainen PV, Arvola T, Salminen SJ, Isolauri E [2002] Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? Gut 51: 51-55.21.
21. Momose H, Igarachi M, Kawashima T, Kuboyama M [1982]The effect of bifidus milk administration on the ecology of Enterobacteriaceaein digestive tract. XXI Int. Dairy Congr, Moscow, Vol. 1, pp. 348.
22. Misra AK, Kuila RK [1994] Effect of bifidus milk feed on excretory pattern of coliforms. Indian J Dairy Sci 47: 603-606.
23. Sarkar S, Misra AK [1998] Effect of feeding Propiono-Acido- Bifido [PAB] milk on the nutritional status and excretory pattern in rats and children. Milchwiss 53: 666-668.
24. Sarkar S, Misra AK [2000] Assessment of nutritional and therapeutic characteristics of Propiono-Acido-Bifido [PAB] milk through rat-bioassay technique. Indian J Dairy Biosci 11: 27-31.
25. Sarkar S, Misra AK [2002] Effect of feeding dietetic yoghurt on the nutritional status and excretory pattern in rats and infants. Egypt J Dairy Sci 30: 63-73.
26. Patel JR, Dave JM, Dave RI, Sannabhadti SS [1992] Effect of feeding milk fermented with mixed culture of human strains of lactobacilli on faecal lactobacilli and coliform counts in human test subjects.Indian J Dairy Sci 45: 379-382.
27. Goldin BR, Gorbach, SL [1984] Alteration of the intestinal microflora by diet, oral antibiotics, and Lactobacillus: decreased production of free amines from aromatic nitro compounds, azo dyes and glucuronides.J Natl Cancer Inst 73: 689-95.
28. Lidbeck A, Geltner Allinger U, Orrhage KM, Ottova L, Brismar B, et al. [1991] Impact of Lactobacillus acidophilus supplements on the faecal microflora and soluble faecal bile acids in colon cancer patients. Microbial Ecol Hlth Dis 4: 81-88.
29. Spanhaak S, Havenaar R, Schaafsma G [1998] The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans. Eur J Clin Nutr 52: 899-907.
30. Marteau P, Flourie B, Pochart P, Chastang C, Desjeux JF, et al. [1990] Effect of the microbial lactase [EC 3.2.1.23] activity in yoghurt onthe intestinal absorption of lactose: an in vivo study in lactase-deficienthumans. Br J Nutr 64: 71-79.
31. Kim HS, Gilliland SE [1983] Lactobacillus acidophilus as a dietary adjunct for milk to aid lactose digestion in humans. J Dairy Sci 66: 959-966.
32. Gilliland SE, Kim HS [1984] Effect of viable starter culture bacteria in yoghurt on lactose utilization in humans. J Dairy Sci 67:1-6.
33. Savaiano DA, Abou El Anouar A, Smith DE, Levitt MD [1984] Lactose malabsorption from yoghurt, pasteurized yoghurt, sweet acidophilus milk and cultured milk in lactase-deficient individuals. Am JClin Nutr 40:1219-1923.
34. Dewit O, Pochart P, Desjeux JF [1988] Breath hydrogen concentration and plasma glucose, insulin and free fatty acid levels after lactose, milk, fresh and heated yoghurt ingestion by healthy young adults with or without lactose malabsorption. Nutr 4: 1-5.
35. Pochart P, Dewit O, Desjeux JF, Bourlioux P [1989] Viable starter culture, -galactosidase activity and lactose in duedenum after yoghurt ingestion in lactase deficient humans. Am J Clin Nutr 49: 828-831.
36. Sarkar S [2002] Nutritional and healthful aspects of cultured milk products - A Review. Indian J Dairying Biosci 13: 1-9.
37. Sarkar S, Misra AK [2002] Yoghurt:Nutritional and therapeutic significance. Indian J Microbiol Vol 42: 275-287.
38. Mijacevic Z, Bulajic S, Nedic D [2001] Survival and therapeutic potential of probiotic microorganisms in fermented milk. Acta Veterinaria51: 325-332.
39. Kurmann JA, Rasic JL [1991] The health potential of products containing bifidobacteria. In: Therapeutic properties of fermented milks,Ed. Robinson, R.K., Elsevier Science Publishers Ltd., London, pp. 117-58.
40. Lourens-Hattingh A, Viljoen, BC [2001] Yoghurt as probiotic carrier food. Int Dairy J 11: 1-17.
41. Pelletier X, Laure-Boussuge S, Donazzolo Y [2001] Hydrogen excretion upon ingestion of dairy products in lactose intolerant male subjects: Importance of the live flora. Eur J Clin Nutr. 55: 509-512.
42. Ross RP, Desmond CC, Fitzgerald GF, Stanton C [2005] Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbiol 98: 1410-1417.
43. Samona A, Robinson RK [1994] Effect of yogurt cultures on the survival of bifidobacteria in fermented milks. J Soc Dairy Technol 47: 58-60.
44. Dinakar P, Mistry VV [1994] Growth and viability of Bifidobacterium bifidum in cheddar cheese. J Dairy Sci 77: 2854-2864.
45. IDF [1992] General standard of identity for fermented milks. Vol. 163 International Dairy Federation, Brussels, Belgium.
46. ANZFA [2003] Fermented milk products. Standard 2.5.3., Australia and New Zealand Food Authority, Canberra, Australia: Australian Government Printing Services.
47. Bibiloni R, Zavaglia AG, Antoni GD [2001] Enzyme-based most probable number method for the enumeration of Bifidobacterium in dairy products. J Fd Protect 64: 2001-2006.
48. Ministerio de la Presidencia de Espano [2003] Norma de Calidad del Yogur. RD 179/2003, BOE 18 de Febrero, 2003.
49. Shah NP, Ravula RR [2000] Influence of water activity on fermentation, organic acids production and viability of yoghurt and probiotic bacteria. Aus J Dairy Technol 55: 127-131.
50. Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, et al. [2003] Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Fd Microbial 82: 59-70.
51. Weese JS [2003] Evaluation of deficiencies in labelling of commercial probiotics. Can Vet J 44: 982-983.
52. Hamilton-Miller JMT, Shah S, Winkler JT [1999] Public health issues arising from microbiological and labeling quality of foods and supplementscontaining probiotic microorganisms. Public Hlth Nutr 2: 223-229.
53. Bersani C, Ripamonti B, Nadini A, Cantini C [2006] Lactic microflora in drinking yoghurt with added probiotics. Ingre Alimentai 5:6-10.
54. Carr JP, Ibrahim SA [2005] Viability of bifidobacteria in commercial yoghurt products in North Carolina. Milchwiss 60: 414-416.
55. Ibrahim SA, Carr JP [2006] Viability of bifidobacteria in commercial yoghurt products in North Carolina during refrigerated storage. Int J Dairy Tech 59: 272-77.
56. Jayamanne VS, Adams MR [2006] Determination of survival, identity and stress resistance of probiotic bifidobacteria in bio-yoghurts. Letters Appl Microbiol 42: 189-194.
57. Perea Velez M, Hermans K, Verhoeven TL, Lebeer SE, Vanderleyden J, et al. [2007] Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products. J ApplMicrobiol 103: 666-674.
58. Iwana H, Masuda H, Fujisawa T, Suzuki H, Mitsuoka T [1993] Isolation and identification of Bifidobacterium spp. in commercial yoghurts sold in Europe. Bifidobacteria Microflora 12: 39-45.
59. Shah NP, Ali JF, Ravula RR [2000] Populations of Lactobacillus acidophilus, Bifidobacterium spp. and Lactobacillus casei in commercial fermented milk products. Biosci Microflora19: 35-39.
60. Schillinger U [1999] Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage. Int J Fd Microbiol 47: 79-87.
61. Rybka S, Fleet GH [1997] Populations of Lactobacillus delbrueckii ssp bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium species in Australian yoghurts. Fd Aus 49:471-475.
62. Shah NP [1997] Bifidobacteria: Characteristics and potential for application in fermented milk products. Milchwiss 52: 16-21.
63. Dave RI, Ramaswamy N, Boer RJ [2002] Changes in fatty acid composition during yoghurt processing and their effects on yoghurt and probiotic bacteria in milk procured from cows fed different diets. Aus J Dairy Technol 57: 197-202.
64. Micanel N, Haynes IN, Playne MJ [1997] Viability of probiotic cultures in commercial Australian yogurts. Aus J Dairy Technol 52: 24-27.
65. Gregurek L [1999] Effect of level of starter culture on viability of probiotic bacteria in yoghurts. Mljekarstvo 49: 3-8.
66. Vinderola CG, Bailo N, Reinheimer JA [2000] Survival of probiotic microflora in Argentinian yoghurts during refrigerated storage. Fd Res Int 33: 97-102.
67. Baig MI, Prasad V [1996] Biochemical characteristics and stability of Bifidobacterium bifidum in frozen yoghurt supplemented with condensed cheese whey. J Dairying Fd Home Sci 15: 99-108.
68. Vinderola CG, Mocchiutti P, Reinheimer JA [2002] Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products. J Dairy Sci 85: 721-729.
69. Talwalkar A, Kailasapathy K [2004] The role of oxygen in the viability of probiotic bacteria with reference to L. acidophilus and Bifidobacterium spp. Curr Issues Intest Microbiol 5: 1-8.
70. Payne JF, Morris AEJ, Beers PJ [1998] Viability of bifidobacteria in fermented milk products”, Functional foods: the consumer, the products and the evidence, Ed. Sadler MJ, Saltmarsh M, pp. 143-148.
71. Mattila-Sandholm T, Myllarinen P, Crittenden R, Mogensen G, Fonden R, et al. [2002] Technological challenges for future probioticfoods. Int Dairy J 12: 173-182.
72. Conway PL, Gorbach SL, Goldin BR [1987] Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J Dairy Sci 70: 1-12.
73. Pedrosa MC, Golner BB, Goldin BR, Barakat S, Dallal GE, et al. [1995] Survival of yogurt-containing organisms and Lactobacillus gasseri [ADH] and their effect on bacterial enzyme activity in the gastrointestinaltract of healthy and hypochlorhydric elderly subjects. Am J ClinNutr 61: 353-359.
74. Bezkorovainy A [2001] Probiotics: determinants of survival and growth in the gut. Am J Clin Nutr 73: 399-405.
75. Sarkar S, Misra AK [2006] Probiotic acidophilus milk for infants and children. Nutr Fd Sci 36: 349-356.
76. Pochart P, Marteau P, Bouhnik T, Goderel I, Bourlioux P, et al. [1992] Survival of bifidobacteria ingested via fermented milk during their passage through the human small intestine: an in vivo study using intestinal perfusion. Am J Clin Nutr 55: 78-80.
77. Marteau P, Minekus M, Havenaar R, Huis in’t Veld JHJ [1997] Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile. J Dairy Sci 80: 1031-1037.
78. Shah NP [2000] Probiotic bacteria: Selective enumeration and survival in dairy foods. J Dairy Sci 83:894-907.
79. Dave RI, Shah NP [1997] Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures. Int Dairy J7: 31-41.
80. Mortazavian AM, Ehsani MR, Mousavi SM, Reinheimer JA, Emamdjomeh Z, Sohrabvandi S, Rezaei K (2006) Preliminary investigation of the combined effect of heat treatment and incubation temperatureon the viability of the probiotic micro-organisms in freshly made yogurt. Int J Dairy Technol 59: 8-11.
81. Kailasapathy K, Godward G [2001] Microencapsulation of probiotic bacteria with alginate starch and evaluation of their survival in dairy products. Proc. Int. Congr Probiotic Med, Biological Medicine Press, 6-8 July, 2001, pp.77-84.
82. Heller KJ [2001] Probiotic bacteria in fermented foods: product characteristics and starter organisms. Am J Clin Nutr 73: 374-379.
83. Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S (2001) Quality assurance criteria for probiotic bacteria: Evaluation of deficiencies in labeling of commercial probiotics. Am J Clin Nutr 73:393-398.
84. BFAD [2004] Guidelines on probiotics. Bureau Circular, No. 16 s. 2004, Department of Health, Filinvest Corporate City, Alabang, Muntinlupa City, Bureau of Food and Drugs, 26 October 2004.
85. Asp N, Mollby R, Norin L, Wadstrom T [2004] Probiotics in gastric and intestinal disorders as functional food and medicine. Scand J Nutr 48: 15-25.
86. Harish K, Varghese T [2006] Probiotics in humans–evidence based review. Calicut Med J 4: 3.
87. Dekker J, Collett M, Prasad J, Gopal P [2007] Functionality of probiotics - potential for product development. Forum Nutr 60: 196-208.
88. CAST (2007) Probiotics: Their potential to impact human health. Issue Paper 36, Council for Agricultural Science and Technology, Ames, Iowa, pp, 1-20.
89. FAO/WHO (2001) Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria”, Report of a Joint FAO/WHO Expert Consultation on Evaluation ofHealth and Nutritional Properties of Probiotics in Food Including PowderMilk with Live Lactic Acid Bacteria Amerian Cordoba Park Hotel,Cordoba, Argentina 1-4 October 2001, Food and Agriculture Organizationof the United Nations World Health Organization. pp. 1-38.
90. Del Piano M, Morelli L, Strozzi GP, Allesina S, Barba M, et al. [2006] Probiotics: from research to consumer. Dig Liver Dis 38: 248-255.
91. Tamboli CP, Caucheteux C, Cortot A, Colombel JF, Desreumaux P [2003] Probiotics in inflammatory bowel disease: a critical review. Best Pract Res Clin Gastroenterol 17: 805-820.
92. Reid G [2005] The Importance of Guidelines in the Development and Application of Probiotics. Current Pharmaceutical Design 11:11-16.
93. Sanders ME [1999] Probiotics. Fd Technol 53: 67-75.
94. FAO/WHO [2002] Guidelines for the Evaluation of Probiotics in Food. Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, London, Ontario, Canada, April 30 and May 1, 2002, Food and Agriculture Organization of the United Nations World Health Organization, pp.39-50.
95. Donohue DC [2006] Safety of probiotics. Asia Pacific J ClinNutr 15: 563-569.