Microbiological Considerations for Probiotic Supplemented Foods
Quality Assurance, Metro Dairy Limited, Barrackpore-Barasat Link Road, Subhasnagar, P.O. Neelgunj Bazar, Kolkata-700121, West Bengal, India
Quality Assurance, Metro Dairy Limited, Barrackpore-Barasat Link Road, Subhasnagar, P.O. Neelgunj Bazar,
Kolkata-700121, West Bengal, India.
Article Type: Research Article
Received: February 20, 2013; Accepted: March 20, 2013; Published: April 19, 2013
Citation: S. Sarkar. (2013). Microbiological Considerations for Probiotic Supplemented Foods. Int J Microbiol Adv Immunol, 1(1), 1-5. doi: dx.doi.org/10.19070/2329-9967-130001
Copyright: S. Sarkar © 2013. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Functional properties of probiotics coupled with consumer’s inclination towards healthful foods have projected probiotics as a new ingredient in functional food market. Probiotic containing foods exhibits diverse health benefits and the starter cultures employed for formulation of probiotic supplemented food must possess certain prerequisite characteristics to exhibit prophylactic properties. Probiotic containing foods available in the market are often of poor quality and did not meet the desired level of viable microorganisms, required for exhibiting health benefits. In the present article an endeavor has been made to highlight the significance of probiotic viability and their population for exhibiting health benefits and the quality of probiotic containing foods available in the global market and prerequisites for identity of a product as a probiotic food have also been delineated. Production of probiotic supplemented food with prophylactic is emerging to build-up consumer’s confidence for long-term sustainability of probiotic food industries.
3.Viability of Probiotic Cultures
3.1. Significance of Probiotic Viability
4. Viability of Probiotics in Probiotic Foods
4.1. Factors Affecting Viability of Probiotics
5.Microbiological Considerations for Probiotic Supplemented Foods
5.1. Microbiological Considerations for Probiotic Selection
5.2. Microbiological Considerations for Health Claims
5.3. Microbiological Considerations for Safety Aspects
Probiotic;Health claims; Food; Starter cultures.
Fermentation employing lactic acid bacteria is the oldest,simple and safest means of food preservation  and relatively recently their efficacy to exhibit health benefits have been explored . 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 .
Probiotics may be defined as “live microbial food ingredient that, when ingested in sufficient quantities, exert health benefits on the consumer” . Probiotics are now emerged as an important category of food supplement and could be found in conventional, dietary supplements and medicinal foods  in many countries including Japan, Europe and USA . Significance of human gut microbiota in health restoration and maintenance have led acceptance of probiotics as functional foods  in current era of self-care and complementary medicine [7, 8] and comprises approximately 65% of the world’s functional food market . Owing to diverse prophylactic properties, probiotic foods could fall into the category of functional foods  and consumption of functional probiotic bacteria is increasing due to promotion of gut health, disease prevention and therapy . Beneficial health effects extend by probiotics is due to maintenance of the equilibrium of indigenous microbiota  with the growth inhibition of pathogenic microorganisms and boosting of innate and acquired immunity . 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  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 . Further well-designed placebo-controlled studies are emerging for determining the optimal dose, duration of treatment, selection probiotic strains, their mode of actions  and efficacy of multi-strain preparations  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
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 . Ingestion of yoghurt containing 108 cfu/g B. longum  and hydrolyzed whey formulae containing 1x109 cfu/g B. lactis  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 . 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 , Propiono-Acido-Bifido [PAB] milk [23, 24] and dietetic yoghurt  containing B. bifidum [108 cfu/ml]. Ingestion of fermented milk containing 5x107 cfu/ml L. acidophilus and L. casei by human volunteers  or bifidus milk containing 108 cfu/g B. bifidum by infants  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  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 .
An improvement in lactose digestion due to ingestion of milk supplemented with L. acidophilus (2.5x106 to 2.5x10,8cfu/ml) was observed . 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 . Decrement in viable population (3x108/g to 3.4x106/g) and lactase activity (0.64 to 0.07 units/g) due to pasteurization  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 . 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 . 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 . The recommended intake is 300-400g/ week  or 100g/day . 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 .
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 . 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 . 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 .
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  and are present at non-detectable levels or at a level of 104 -107 cfu/ml  or 106 cfu/ml . Bifidobacteria were less acid tolerant than L. acidophilus  and were detected at a level of 106 cfu/ml, respectively in 14 and 24% of yoghurt samples  and both retained their viability at a level of >105cfu/ml during storage . Lower bifidobacterial population (< 103 cfu/g)than L. acidophilus (< 103-108 cfu/g) were detected in few Australian yoghurt containing probiotic cultures . 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 . 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 . 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 . 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  due to loss of viability by heat, pressure, low water activity and high acidity .
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  and must be adapted to the intestinal environment for its prolonged survival , as only 20-40% probiotic cultures survive the gastric transit . Though appreciable growth of B. bifidum and L. acidophilus in presence of bile salts  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 . It has been annunciated that ingestion of fermented milk containing probiotic cultures resulted in survival of 23.5±10.4% bifidobacteria , 30% B. bifidum, 10% L. acidophilus , 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.
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 
• concentration of lactic and acetic acids in the product .
• fat content of milk 
• heat-treatment of milk
• incubation temperature 
• concentration of buffers such as whey protein concentrate 
• physiological status of probiotic cultures added
• physical condition of product storage
• possible interactions of the product with starter cultures.
Microbiological Considerations for Probiotic Supplemented Foods,
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 
• beneficial effect on the host organism
• should adhere to the mucosal epithelial cells
• should exhibit enhancement and protection of the intestinal ecology 
• does not have the ability to invade the host intestinal tissues and cause any infection
• sensitive to broad spectrum and commonly used antibiotics
• should be isolated from the same species as its intended host
• should be able to survive transit through the gastrointestinal tract 
• every strain must exhibit efficacy of health benefits 
• 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 
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 . 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 . 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 . Various clinically relevant steps required for the acceptance of probiotics by the medical community are enumerated underneath .
• 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
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 . 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 . 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 
• 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 
• 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 
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.
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