Probiotics notes
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This document provides an overview of intestinal beneficial bacteria. It discusses the microbiology of the human gastrointestinal tract, important characteristics of beneficial bacteria, and their probiotic effects such as lactose hydrolysis, reducing serum cholesterol and colon cancer, and modulating immune response. Some considerations for probiotic use are addressed, like strain variation and sensitivity to stomach acids. Current developments discussed include standardization, the scientific status of probiotics, and synergistic combinations of probiotics and prebiotics.
Probiotics notes
- 1. Table of Contents Section I Introduction to Microbes in Foods ......................................................1 Chapter 1 History and Development of Food Microbiology ...................................................3 Chapter 2 Characteristics of Predominant Microorganisms in Food .....................................13 Chapter 3 Sources of Microorganisms in Foods ....................................................................35 Chapter 4 Normal Microbiological Quality of Foods and its Significance ........................... 43 Section II Microbial Growth Response in the Food Environment.................... 55 Chapter 5 Microbial Growth Characteristics ..........................................................................57 Chapter 6 Factors Influencing Microbial Growth in Food .....................................................67 Chapter 7 Microbial Metabolism of Food Components .........................................................81 Chapter 8 Microbial Sporulation and Germination ................................................................93 Chapter 9 Microbial Stress Response in the Food Environment .........................................103 Section III Beneficial Uses of Microorganisms in Food ..................................123 Chapter 10 Microorganisms Used in Food Fermentation ......................................................125
- 2. 209 CHAPTER 15 Intestinal Beneficial Bacteria CONTENTS I. Introduction ..............................................................................................210 II. Microbiology of the Human GI Tract ......................................................211 III. Important Characteristics of Beneficial Bacteria .....................................212 IV. Beneficial Effects of Probiotics ................................................................213 A. Lactose Hydrolysis1,4–6 ..................................................................213 B. Reducing Serum Cholesterol Level1,4–6 ........................................214 C. Reducing Colon Cancer1,4–6 ..........................................................214 D. Reducing Intestinal Disorders1,4–6 .................................................214 E. Modulating Immune Response1,4–7 ...............................................215 F. Reducing Allergic Diseases1,4–7 ....................................................215 G. Miscellaneous Benefits ....................................................................216 V. Some Aspects to Consider .......................................................................216 A. Strain Variation .................................................................................216 B. Sensitivity to Stomach Acids ...........................................................217 C. Viability and Injury of Cells ............................................................217 D. Dose Level and Duration .................................................................217 E. Induced Lactase Trait .......................................................................218 F. Antibacterial Substances ..................................................................218 G. True Species and Strains ..................................................................218 H. Expertise in Research Areas ............................................................218 VI. Current Developments ..............................................................................218 A. Standard of Identity .........................................................................219 B. Scientific Status Summary ...............................................................219 C. Pathogenic Nature .............................................................................219 D. Probiotics, Prebiotics, and Synbiotics .............................................220 1. Probiotics ....................................................................................220 2. Prebiotics ....................................................................................221 3. Synbiotics ...................................................................................221
- 3. 210 FUNDAMENTAL FOOD MICROBIOLOGY E. Biogenics ..........................................................................................221 F. Genome Sequence of Probiotic Bacteria..........................................222 VII. Conclusion ................................................................................................222 References ............................................................................................................222 Questions ..............................................................................................................223 I. INTRODUCTION Since the discovery of food fermentation, our ancestors recognized that the process yielded products that had not only better shelf life and desirable qualities but also some health benefits, especially to combat some intestinal ailments. The belief in the health benefits of fermented foods continued throughout civilization and even today remains of interest among many consumers and researchers.1 There are dif- ferences between early beliefs and the current interest: whereas the early beliefs probably emerged from associated effects (benefit from consuming) without know- ing the scientific basis, current interest is based on understanding the microbiology and biochemistry of fermented foods, microbial ecology of the human gastrointes- tinal (GI) tract, and roles of some bacteria in the GI tract and in fermented foods in human health. Researchers, however, are divided in their opinions. Some advocate different health benefits that consumers can have from consuming fermented foods, especially fermented dairy products, but others doubt those attributes, especially when "cure-all" claims are made by overzealous individuals with none or very little scientific background in the area. In Western countries, the beneficial role of fermented milk (yogurt) in the prolongation of life was first advocated by Metchnikoff in 1907. He suggested that the bacteria and their metabolites in yogurt neutralized the harmful products yielded from foods in the GI tract and provided protection to human health. However, later studies produced controversial results. In recent years, especially since the 1970s, consumers in many developed coun- tries have been particularly interested in the health benefits of foods. There is an increased interest in foods that are not harshly processed and preserved, but are natural, and fermented foods are considered natural and healthy. Consumers' interest in and demand for fermented foods have resulted in a large production increase of many products that had very small markets before. Many ethnic products have earned commercial status. In addition, consumers’ interest in the health benefits of some bacterial species has stimulated production of new products containing live cells of these bacteria. These products are generally designated as probiotics. The term has been used loosely for a while, but is currently defined as a product containing living microorganisms, which on ingestions in certain numbers exert health benefits beyond inherent general nutrition.2 This trend will continue not only in the developed countries but also in many developing countries, particularly where fermented foods have been consumed for a long period of time. However, it will be extremely important to resolve the present controversies on the health benefits of probiotics, which can only be possible by understanding the current problems, conducting well-designed experiments, and
- 4. INTESTINAL BENEFICIAL BACTERIA 211 analysing the results scientifically. In this chapter, the current status of our knowledge on intestinal microbial ecology, beneficial microorganisms of the GI tract, beneficial effects of fermented milk products, and the probable causes of some controversies are briefly presented. II. MICROBIOLOGY OF THE HUMAN GI TRACT The GI tract of humans contains more than 1014 microorganisms, many more than the total number of our body cells. They are metabolically diverse and active; thus it is quite likely that they have a great influence on our well-being. It is estimated that the human GI tract harbors ca. 1000 bacterial species, but only 30 to 40 species constitute 95% of the population.2 Normally, the microbial level in the small intestine (particularly in the jejunum and ilium) is ca. 106–7/g, and in the large intestine (colon) ca. 109–10/g of the content. The most predominant types in the small intestine are several species of Lactobacillus and Enterococcus, and in the large intestine are several genera of Enterobacteriaceae, different species of Bacteroides, Fusobacte- rium, Clostridium, Eubacterium, Enterococcus, Bifidobacterium, and Lactobacillus.3 The intestine of a fetus in the uterus is sterile. At birth, it is inoculated with vaginal and fecal flora from the mother. Subsequently, a large variety of microor- ganisms enter in the digestive tract of infants from the environment. From these, the normal flora of the GI tract are established. In both breast-fed and formula-fed babies during the first couple of days, Escherichia coli and Enterococcus appear in large numbers in the feces. Then, in the breast-fed babies, large numbers of Bifidobacte- rium species and a lower level of both Esc. coli and Enterococcus species appear. In formula-fed babies, in contrast, Esc. coli and Enterococcus, together with Clostridium and Bacteroides, predominate, with Bifidobacterium being almost absent; this situation may lead to diarrhea. As breast-fed babies are introduced to other foods, the levels of Esc. coli, Enterococcus, Bacteroides, Clostridium, and others increase, but Bifidobacterium still remains high. When breast-feeding is completely stopped, Bacteroides, Bifidobacterium, and Lactobacillus species pre- dominate, along with some Esc. coli, Enterococcus, Clostridium, and others. By the second year of life, the different microflora establish themselves at their specific ecological niche in the GI tract, and the population resembles that of adult GI tracts. The intestinal microflora are divided into indigenous (autochthonous) and tran- sient (allochthonous) types. Many indigenous species can adhere to intestinal cells, which helps maintain them in their specific niche. Whereas the indigenous types are permanent inhabitants, the transient types are either passing through or temporarily colonizing a site from where the specific indigenous type has been removed because of some inherent or environmental factors (such as antibiotic intake). Among the indigenous microbial flora, several species of Lactobacillus in the jejunum and ilium, and Bifidobacterium in the large intestine, are thought to have beneficial effects on the health of the GI tracts of the hosts. From the intestines and intestinal content of humans, Lactobacillus acidophilus, Lab. fermentum, Lab. rha- monosus, Lab. reuteri, Lab. casei, Lab. lactis, Lab leichmannii, Lab. plantarum, Bifi- dobacterium bifidus, Bif. longum, Bif. adolescentis, Bif. infantis, and others have been
- 5. 212 FUNDAMENTAL FOOD MICROBIOLOGY isolated. However, age, food habits, and health conditions greatly influence the species and their levels. There is some belief that a portion of the intestinal Lactobacillus species is transient. Initially, it was considered that Lab. acidophilus, Lab. reuteri, and some Bifidobacterium are the main indigenous species. At present, several other Lac- tobacillus species, such as Lab. casei and Lab. rhamnosus, have been tested to be beneficial. The presence of high numbers of the indigenous Lactobacillus species in the feces (and content of the large intestine) probably results from their constant removal from the small intestine. III. IMPORTANT CHARACTERISTICS OF BENEFICIAL BACTERIA Some relevant characteristics of Lab. acidophilus, Lab. reuteri, and Bifidobacterium species are briefly discussed here.4–6 All three are found in the GI tract of humans as well as in animals and birds. They are Gram-positive rods and grow under anaerobic conditions. Lab. acidophilus is an obligatory homolactic fermentator; Lab. reuteri is a heterolactic fermentator and produces lactic acid, ethanol, and CO2; and Bifidobacterium species produce lactic and acetic acids (in 2:3 ratio). They are less sensitive to stomach acid than many other bacteria under a given condition, and highly resistant to bile, lysozyme, and pancreatic enzymes present in the GI tract (small intestine). The two Lactobacillus species are present in low numbers in the jejunum, but in relatively high numbers in the ilium, especially toward the distal part, whereas Bifidobacterium species are present in the proximal part of the colon (near the ilium). All three are able to colonize in their respective niches, but studies with Lab. acidophilus reveal that all strains do not adhere to the GI mucosa of the host. Other studies have shown that the ability to adhere to the intestinal epithelial cells by Lab. acidophilus strains can be species specific, i.e., a specific strain can adhere to a particular species only and the trait may be lost during prolonged culturing under laboratory conditions. Under normal conditions, these three species are thought to help in maintaining the ecological balance of GI tract microflora by controlling growth rate of undesirable microflora. This effect is produced through their ability to metabolize relatively large amounts of lactic and acetic acids. In addition, they can produce specific inhibitory substances, several of which are recognized. Many strains of Lab. acidophilus are known to produce bacteriocins, although they are most effective against closely related Gram-positive bacteria. Also, because of the sensitivity of bacteriocins to proteolytic enzymes of the GI tract, their actual role in controlling undesirable Gram-positive bacteria in the GI tract is disputable. Some strains also produce compounds that are not well characterized, but have been reported to have an antibacterial effect on both Gram-positive and Gram-negative bacteria. Some Lab. reuteri strains while growing in glycerol produce reuterine, which is inhibitory to both Gram-positive and Gram- negative bacteria.Antibacterial metabolite production by a few Bifidobacterium species is also reported. Some strains of Lab. acidophilus are able to deconjugate bile acids to produce compounds that are more inhibitory than the normal bile acids. Some Lactobacillus strains can also produce H2O2, but probably not under anaerobic condi- tions in the GI tract. Identification of specific antibacterial compounds other than acids
- 6. INTESTINAL BENEFICIAL BACTERIA 213 (such as reuterine) in these beneficial bacteria will help in understanding their role in maintaining intestinal health. Several studies have indicated that beneficial effects of these bacteria are produced when they are present in relatively high numbers in the intestinal tract ( 106–7/g intestinal content). Diets rich in foods from plant sources, as opposed to those rich in foods from animal sources, seem to favor their presence in higher numbers. Many other conditions in a host also can reduce bacterial numbers in the GI tract, such as antibiotic intake, mental stress, starvation, improper dietary habits, alcohol abuse, and sickness and surgery of the GI tract. This, in turn, can allow the undesirable indigenous or transient bacteria to grow to high levels and produce enteric disturbances, including diarrhea, flatulence, and infection by enteric pathogens. IV. BENEFICIAL EFFECTS OF PROBIOTICS In the last 40 years, studies have been conducted to determine specific health benefits from the consumption of live cells of beneficial bacteria.1,4–7 Live cells have been consumed from three principal sources: (1) as fermented milk products, such as yogurt, which contains live cells of Lab. delbrueckii ssp. bulgaricus and Str. ther- mophilus and is supplemented with Lab. acidophilus and others, and pasteurized milk, which contains Lab. acidophilus; (2) as supplementation of foods and drinks with live cells of one, two, or more types of beneficial intestinal bacteria, such as Lab. acidophilus, Lab. reuteri, Lab. casei, and Bifidobacterium species; and (3) as pharmaceutical products of live cells in the form of tablets, capsules, and granules. The beneficial effects from consuming these live cells were attributed to their ability to provide protection against enteric pathogens, supply enzymes to help metabolize some food nutrients (such as lactase to hydrolyze lactose) and detoxify some harmful food components and metabolites in the intestine, stimulate intestinal immune sys- tems, and improve intestinal peristaltic activity. Some of these are briefly discussed here. In addition, many other benefits have been claimed by entrepreneurs, without any scientific basis, and they are not discussed. A. Lactose Hydrolysis1,4–6 Lactose-intolerant individuals, because of a genetic disorder, are unable to produce lactase (b-galactosidase) in the small intestine. When they consume milk, lactose molecules are not hydrolyzed in or absorbed from the small intestine but passed to the colon. They are then hydrolyzed in the colon by lactase of different bacteria to glucose and galactose and then further metabolized to produce acids and gas, result- ing in fluid accumulation, diarrhea, and flatulence. Consumption of yogurt, acido- philus milk, live cells of Lactobacillus, especially Lab. acidophilus in fresh milk and pharmaceutical products, reduces the symptoms in lactose-intolerant individuals. This benefit is attributed to the ability of beneficial bacteria to supply the needed lactase in the small intestine. However, as Lab. delbrueckii ssp. bulgaricus and Str. thermophilus do not survive stomach acidity well and are not normal intestinal bacteria, the benefit of consuming normal yogurt is considered to be due to the
- 7. 214 FUNDAMENTAL FOOD MICROBIOLOGY reduced amounts of lactose in yogurt, as compared to milk, and to the supply of lactase from the dead cells. In contrast, the intestinal bacteria, especially some Lactobacillus species, could, under proper conditions, colonize the small intestine and subsequently supply lactase. Different studies, however, did not unequivocally prove the desired benefits, probably because of differences in study methods, includ- ing the use of strains that lack b-galactosidase, use of strains that are not host specific, use of species that are not of the intestinal type or unable to adhere to the intestine, use of preparations with low viable cells, and lack of expertise in microbiology and gastroenterology research. B. Reducing Serum Cholesterol Level1,4–6 Consumption of fermented dairy products (some containing unknown microor- ganisms) and high numbers of live cells of beneficial intestinal bacteria has been associated with low levels of serum cholesterol in humans. This is attributed to two possible factors. One is the ability of some intestinal lactobacilli to metabolize dietary cholesterol, thereby reducing amounts absorbed in blood. The other pos- sibility is that some lactobacilli can deconjugate bile salts and prevent their reabsorption in the liver. The liver, in turn, uses more serum cholesterol to syn- thesize bile salts and indirectly helps reduce cholesterol level in serum. However, results of several studies by different researchers do not always favor this hypoth- esis. Again, this can be due to differences in experimental design, such as use of strains that do not metabolize cholesterol or deconjugate bile acid, and other reasons described before. C. Reducing Colon Cancer1,4–6 Many of the undesirable bacteria in the colon have enzymes that can activate procarcinogens, either present in food or produced through metabolism of unde- sirable bacteria, to active carcinogens that, in turn, can cause colon cancer. Beneficial intestinal bacteria, both Lactobacillus and Bifidobacterium species, by their ability to control growth of undesirable bacteria in the colon, can reduce the production of these enzymes. Also, beneficial bacteria, by increasing intestinal peristaltic activity, aid in regular removal of fecal materials. This, in turn, lowers the concentrations of the enzymes and carcinogens in the colon and reduces the incidence of colon cancer. Several studies have shown that oral consumption of large numbers of live cells of the beneficial bacteria reduces fecal concentrations of enzymes such as b-glucuronidase, azoreductase, and nitroreductase of unde- sirable colon bacteria. However, the relationship between reduction of these enzymes and reduction in colon cancer from the consumption of beneficial intes- tinal bacteria has not been studied, and thus contributions of these bacteria in controlling colon cancer are not clearly known. Animal studies have shown that formation of aberrant crypts, considered to be putative precancerous lesions, is reduced by consuming live cells of beneficial GI tract bacteria, especially bifodo- bacteria. Again, the results are not consistent, which could be due to the factors listed before.
- 8. INTESTINAL BENEFICIAL BACTERIA 215 D. Reducing Intestinal Disorders1,4–6 Under certain conditions, indicated before, the intestinal population of beneficial bacteria can be reduced. The undesirable bacteria in the intestine and some tran- sient pathogens (such as enteric pathogenic bacteria and Rotoviruses) from the environment can then cause enteric disorders, including infection. Infection results from the invasion and growth of enteric pathogens in the intestine. Ingestion of large numbers of live cells of beneficial intestinal bacteria over a period of time was reported to reduce these problems. Both infants and adults on oral antibiotic therapy can develop diarrhea because of a loss of desirable bacteria in the intestine and an increase in undesirable pathogenic bacteria and viruses. It was suggested that beneficial bacteria, when consumed in large numbers, establish in the intestine and produce antibacterial compounds (acids, bacteriocins, reuterine, and others unknown), which, in turn, control the pathogens. Deconjugation of biles by ben- eficial species also produces compounds that are more antibacterial than the bile salts; this has also been suggested as a mechanism to control the growth of undesirable enteric bacteria. Probiotic bacteria also increase the specific immuno- globulins, reduce intestinal permeability, and normalize intestinal microflora. In many studies, the results were not always positive. This, again, could be due to the variation in study methods, as described before, including the use of strains without the specific trait. E. Modulating Immune Response1,4–7 Limited studies have shown that intestinal microorganisms act on intestinal defense barriers and help regulate systemic and local immune response. This is more effective at an early age, during the development of lymphoid tissues in the gut. Normal establishment of GI tract flora at an early age helps develop immunity to oral administration of antigens associated with inflammatory reaction in the gut. Oral administration of probiotic gut flora also helps overcome some immune response caused by the undesirable gut microflora. This beneficial effect is produced possibly by changing intestinal permeability, altering gut microbiology, improving intestinal immunological barrier functions, and alleviating intestinal inflammatory response. These beneficial effects have not been observed in many other studies. Differences in experimental design, experimental methods, and analysis of the data can be the reasons for the controversy. F. Reducing Allergic Diseases1,4–7 Establishment of normal gut flora, which starts after birth and continues up to two years of age, may be important in the development in later life of counterregulatory ability against several specific immune responses. The normal flora of the GI tract enter the body through food, water, air, and other environmental sources. Raising infants in an oversanitary environment and feeding semisterile processed foods may interfere with the establishment of normal microflora in the GI tract. This may cause the immune system of infants to develop inflammatory response to many food
- 9. 216 FUNDAMENTAL FOOD MICROBIOLOGY antigens. Probiotics containing beneficial gut bacteria can have a suppressive effect to such reaction by stimulating the production of antiinflammatory cytokines and reducing allergic reaction in sensitive individuals. As the results are not consistent, this at present is regarded as only a theory. G. Miscellaneous Benefits Many other health benefits of probiotics have been claimed, such as prophylaxis against urinogenital infection, increased calcium absorption from the intestine, stimulation of endocrine systems, growth promotion, and prolongation of youth and life. Many of these are merely claims and are yet to be proven by proper scientific research. V. SOME ASPECTS TO CONSIDER The health benefit theory of fermented foods and beneficial intestinal bacteria is controversial. Although an association effect, i.e., some benefits from their consump- tion, cannot be denied, many studies have not been able to prove the benefits without doubts. As suggested before, the differences in study methods could be one reason for the differences in results. In designing these studies, several aspects have to be recognized. There are definitely differences in human responses, but there are also differences in bacterial responses. In selecting bacterial strains, the following con- siderations are important.8–11 A. Strain Variation Beneficial strains differ in adherence ability and specificity (Figure 15.1). An adherent strain should probably be favored over a nonadherent strain. Also, strains adherent to humans should be preferred over strains adherent to other species. The selected strains should have a strong adherence property. The adherent property can be lost during long maintenance under laboratory conditions. Many studies have been conducted without even knowing the source and identity of the strains. In selecting a strain for a study, the following factors need to be consid- ered. B. Sensitivity to Stomach Acids Survivability of strains to low stomach pH varies greatly. This effect can be reduced by either reducing stomach acidity with food or by using strains that are proven resistant to acid environment. C. Viability and Injury of Cells Cells of beneficial bacteria when frozen, dried, exposed to low pH, high salts, and many chemicals can die. Among the survivors, many can be injured and killed by
- 10. INTESTINAL BENEFICIAL BACTERIA 217 stomach acid and bile salts and lysozyme in the intestine. In studies, it may be better to use cells grown for 16 to 18 h and maintained before feeding under conditions that retain their maximum viability. D. Dose Level and Duration Consumption of large numbers of live cells (109 per day), that are not stressed, over a period of time (ca. 14 d) is advocated to obtain benefit. Use of preparations that have low levels of viable cells, many of which could be stressed, cannot provide expected results. Many products that are currently marketed do not have the needed level of viable cells to produce beneficial effects (Table 15.1). Some products also have bacteria that are associated with improper sanitary practices. E. Induced Lactase Trait In Lab. acidophilus, lactase is an induced enzyme. To study the lactase effect, strains should be grown in lactose-containing media. In commercial preparations, a strain may be grown in glucose and thus not have lactase when consumed. F. Antibacterial Substances Many studies have reported that strains of beneficial intestinal bacteria produce metabolites that are active against many Gram-positive and Gram-negative bacteria. Some of these were identified, such as several bacteriocins, organic acid, and reu- terine. Other substances need to be identified and examined in purified form for their antibacterial effectiveness. G. True Species and Strains Many species and strains used by many probiotic food producers do not have either proper identity or information of original sources. Also, many species that were previously regarded as Lab. acidophilus have been found to be different species, Figure 15.1 (A) Cells of an adherent strain of Lab. acidophilus are associated with the calf intestinal epithelial cells. (B) Cells of a nonadherent strain of Lab. acidophilus remain uniformly distributed and do not show adherence to epithelial cells from the same source.
- 11. 218 FUNDAMENTAL FOOD MICROBIOLOGY and many are not of intestinal origin. Before selecting a strain for a study, one needs to be sure, through testing by recommended methods, that the strain being used is what it is supposed to be. H. Expertise in Research Areas Lack of an understanding in research in the areas of microbiology, gastroenterology, immunology, oncology, and related fields may result in faulty experimental design and interpretation of data. In addition, differences in response by humans and animals in feeding trials can produce data of little value. Such studies are not expected to produce reproducible results. Research conducted by considering these factors will help reduce bacterial vari- ability. This, in turn, will help compare results of different studies and determine whether the health benefits of these bacteria are real or imaginary. VI. CURRENT DEVELOPMENTS Diverse types of probiotic products are now available in the market, and their consumption has gained popularity, especially in some European and South East Asian countries.Although a long list of health benefits are claimed by manufacturers, most have not been authenticated by well-designed scientific studies. In addition, as mentioned before, many products do not use species and strains that have been Table 15.1 Viable Cell Counts (CFUs) of Lab. acidophilus in Commercial Probiotic Products Product Type CFUs/ga BeneficialbClaimed Actual Health Products (Dry) H1 Not specified 1.5 ¥ 103 c No H2 1 ¥ 108 1.6 ¥ 103 c No H3 4 ¥ 108 7.3 ¥ 102 c No H4 Not specified 2.5 ¥ 106 c No Pharmaceutical Products (Dry) P1 Not specified 4.8 ¥ 106 Maybe P2 Not specified 1.3 ¥ 108 Yes P3 5 ¥ 109 3.2 ¥ 109 Yes P4 2 ¥ 107 2.2 ¥ 103 No a Three samples from separate batches for each product type were enumerated for CFUs/ml in MRS-agar supplemented with 0.15% oxgall. Average results are presented. (From Marteau, P. and Rambaud, J.-E., FEMS Microbiol. Rev., 12, 207, 1993.) b A strain, with proven record of benefit, needs to be consumed at 108–9 viable cells/day for the expected results. On that basis only, consumption of P2 and P3 (provided the strains are beneficial types) can be expected to produce benefit. c The samples had (differed with products) coliform, lactose-negative Gram-negative rods, Bacillus spp., and cocci. This suggests unsanitary practices in the production of these products.
- 12. INTESTINAL BENEFICIAL BACTERIA 219 studied for the possible beneficial effects and many products do not have high viable cell numbers required to produce benefit (Table 15.1). As a result, doubts about the beneficial effect of these products remain. A. Standard of Identity12 Recently, there has been a concerted effort to develop a scientific basis for probiotic bacteria. Through a joint effort by scientists in this area, a standard of identity has been established for the beneficial probiotic bacteria, especially those from the genus Lactobacillus. This includes the ability to (1) adhere to GI epithelial cells; (2) interfere with adherence of enteric pathogens to GI epithelial cells; (3) persist and multiply in the GI tract without disturbing the normal microbial balance; (d) produce antibacterial metabolites; (e) coaggregate; (f) form a normal flora of the GI tract; and (g) be safe to the hosts. Following a critical evaluation of the data of scientific studies, five strains of lactobacilli are considered effective for use as probiotics: (1) Lab. acidophilus NCFM, (2) Lab. casei shirota, (3) Lab. casei CRL431, (4) Lab. rhamonosus GG, and (5) Lab. reuteri MM53. In the last few years, several other species/strains of Lactobacillus and Bifidobacterium have been included in the list. B. Scientific Status Summary13 A scientific status summary has recently been published, listing the diverse species currently being used by probiotic product manufacturers and their claims for the bacterial species they are using. Some of the species include Lab. acidophilus, Lab. bulgaricus, Lab. casei, Lab. fermentum, Lab. johnsonii, Lab. lactis, Lab. paracasei, Lab. plantarum, Lab. rhamonosus, Lab. reuteri, Bif. bifidus, Bif. lon- gum, Bif. brevis, Streptococcus thermophilus, and some yeasts. Their claims are not supported by valid scientific studies and have created disbelief among con- sumers. It was suggested in the report that, to overcome the current controversy, future research should be directed toward (1) identifying the phylogeny of a strain, (2) conducting health benefit studies with humans, (3) explaining the exact mech- anisms by which a strain produces a benefit, (4) finding the dose level of viable cells and duration required for a benefit; (5) determining the ability of a strain to adhere to GI epithelial cells; (6) studying the influence of a probiotic strain on normal GI tract microflora, and (7) determining the proper and effective delivery systems of a probiotic strain. There is no doubt that some bacterial species and strains do have beneficial influence on the health of the GI tract and probably the overall health of humans. But they have to be identified through valid scientific studies. The indiscriminate use, imaginative claims, and unscrupulous methods of a few have created the dis- belief. A move for current collaborative scientific studies to define the standards of identity of probiotic strains, as well as the suggestions outlined in the status report regarding the research requirements before claiming a strain to have benefits, are the correct steps to remove the myths surrounding beneficial properties of probiotic bacteria.
- 13. 220 FUNDAMENTAL FOOD MICROBIOLOGY C. Pathogenic Nature In recent years, there have been a few reports on the isolation of lactic acid bacteria, such as Pediococcus acidilactici and Lab. rhamonosus strains, from infections in humans. Lactic acid bacteria, especially those used in food fermentation and as probiotics, are considered food grade and have been given the GRAS (generally regarded as safe) status internationally. In this regard, involvement of these bacteria in health hazards raised questions about their safety. The best answer to this doubt is an aphorism of the 16th-century German scientist Paracelsus: “All substances are poison — the right dose differentiates a poison and a remedy.” From ancient civi- lization on, people have been consuming live cells of many of these bacteria through foods, without having major incidence of a health hazard, and they, without being eliminated, remained in our food chain. There are three possible reasons for the infections that result: 1. True identity of a strain is not known. Many strains currently used, especially as probiotics, have not been correctly identified to the species level (especially by genetic techniques), have not been tested for their beneficial properties to humans, or their sources have not been identified. The products may harbor pathogens because they have been produced under unsanitary conditions. 2. Being overly anxious for the benefit, an individual may consume a product in large volume. If the individual is immunocompromised, the large dose can cause a health hazard, not as a primary cause but maybe as a secondary cause. 3. Many isolates from infections have not been identified correctly to genus and species level by modern genetic techniques. By fermentation pattern only, it is difficult to identify the genus and species of an isolate in many situations. The incidence of health hazard from beneficial bacteria, even with all the abuses, is very low. If we consider other beneficial things, such as use of antibiotics in the treatment of diseases, the incidence of health risk is much higher. Considering this, it is justifiable to assume that true food-grade lactic acid bacteria are safe. D. Probiotics, Prebiotics, and Synbiotics6,14 1. Probiotics The term probiotics was initially used with no specific definition. In 1989, Fuller2 defined the term as products containing living microorganisms, which on ingestion (by humans, animals, and birds) in certain numbers exert health benefits beyond inherent general nutrition. In this statement, several requirements were included, such as the microbial cells should be alive and consumed in high numbers (usually 109 cells/d). But it did not include which microorganisms (only intestinal or others) should be used and consumed for how long (daily as a preventative or for 2 or more weeks as a curative). It indicates that the consumption should produce health benefit as judged by scientific clinical studies by reputed and independent research groups and results published in peer-reviewed journals (not just a claim by somebody). It also does not specify that the mechanisms by which the health benefits are produced
- 14. INTESTINAL BENEFICIAL BACTERIA 221 should be explained. In the status summary, as discussed before, these aspects have been specifically included. In recent years, several studies have shown that some specific health benefits can be achieved by also consuming products containing dead cells or cell components of beneficial bacteria (e.g., immune modulation). It has to be decided in the future whether they also constitute probiotics. 2. Prebiotics The beneficial effect of probiotic bacteria depends on their presence in high numbers in the GI tract. This can be achieved either by consuming a large number of viable cells of probiotic bacteria or by stimulating rapid growth of desirable gut bacteria by supplying appropriate nutrients. Among the beneficial gut bacteria, Lactobacillus species are predominant in the small intestine whereas Bifidobacterium species are predominant in the large intestine (colon). An approach has been taken to stimulate growth of Bifidobacterium in the colon by supplying one or more selective carbon and energy sources that are not metabolized by the bacteria in the small intestine as well as by many bacteria found in the colon. This gives Bifidobacterium a selective growth advantage and allows it to reach high numbers. These nutrients are termed prebiotics and defined as nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth or activity, or both, of one or a limited number of bacteria in the colon, that can improve the host’s health. Some of the nutrients that have gained importance as prebiotics are lactulose, lactitol, fructooli- gosaccharides, galactooligosaccharides, lactosucrose, and a-inulin. Their actual effectiveness is currently being studied. 3. Synbiotics The term synbiotic is coined to include both the terms probiotics and prebiotics. It is assumed that instead of using probiotics and prebiotics separately, a product containing both, i.e., the beneficial gut bacteria in high numbers as well as nutrient supplement for them will enable them to multiply rapidly in the gut and produce health benefit more effectively. This has to be studied to determine their actual effectiveness. E. Biogenics15 The health benefit from the consumption of fermented dairy products is attributed to the lactic acid bacteria used in fermentation as well as the by-products of metab- olism of milk nutrients by them. The latter aspect becomes important to explain health benefits attributed to fermented products in which nongut bacteria, such as Lactococcus lactis, Str. thermophilus, and Lab. delbrueckii ssp. bulgaricus, are used in fermentation. These products have recently been termed biogenics and include components in food that are derived through the microbial metabolic activity of food nutrients and have health benefits. One such group is the peptide produced by the exoproteinases of some lactic acid bacteria, such as Lac. lactis in buttermilk and
- 15. 222 FUNDAMENTAL FOOD MICROBIOLOGY Lab. delbrueckii ssp. bulgaricus in yogurt. Some of the peptides thus produced and present in the fermented milk can reduce blood pressure in individuals with hyper- tension. This aspect is currently being studied. F. Genome Sequence of Probiotic Bacteria16 The bacterial species and strains that are being studied for genome sequence include several Lactobacillus and Bifidobacterium species that are currently used as probiotic bacteria. These include Lab. acidophilus, johnsonii, casei, and rhamnosus, and Bif. longum, breve and linens. Once the complete genome sequences are obtained, they can be used to compare the genetic and functional diversities of the GI tract beneficial bacteria. This information can then be used in the future to better understand signaling processes among gut microbes, intestinal cells, food components, microbial metabolic products, and mechanisms of beneficial actions of probiotic bacteria. Finally, they will help design a better desirable gut bacteria or specific strains for specific beneficial effects. VII. CONCLUSION Although fermented foods and some lactic acid bacteria have been considered to help to normalize intestinal microbial ecosystems, there are doubts due to differences in results. These are due to improper uses of bacteria that have not been tested by well-designed studies by well-experienced research groups. Currently, standards of identity of beneficial intestinal bacteria are being advocated to change the myths to reality. In the future, genome sequence information will help better understand the interactions of food, intestinal systems, and microbial species and strains and use them effectively. REFERENCES 1. Ouwehand, A.C., Salminen, S., and Isolauri, E., Probiotics: an overview of beneficial effects, Ant. van Leeuwen., 82, 279, 2002. 2. Fuller, R., Probiotics in man and animals, J. Appl. Bacteriol., 66, 365, 1989. 3. Darsar, B.S. and Barrow, P.A., Intestinal microbiology, Am. Soc. Microbiol., Wash- ington, D.C., 1985. 4. Guarner, F. and Schaafsma, G.J., Probiotics, Int. J. Food Microbiol., 39, 237, 1998. 5. Lee, Y.-K., Nomoto, K., Salminen, S., and Gorbach, S.L., Handbook of Probiotics, John Wiley & Sons, New York, 1999. 6. Tannock, G.W., Probiotics: A Critical Review, Horizon Scientific Press, Wymondham, U.K., 2002. 7. Marteau, P. and Rambaud, J.-E., Potential of using lactic acid bacteria for therapy and immunomodulation in man, FEMS Microbiol. Rev., 12, 207, 1993. 8. Brennan, M., Wanismail, B., and Ray, B., Prevalence of viable Lactobacillus acido- philus in dried commercial products, J. Food Prot., 46, 887, 1983.