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Safety of Food and Beverages: Safety of Probiotics and Prebiotics
SAFETY OF FOOD AND BEVERAGES
Safety of Probiotics and Prebiotics UK Svensson, UKS Life Science Consulting AB, Lund, Sweden; Lund University, Lund, Sweden J Ha˚kansson, ASIC, Arla Foods, Stockholm, Sweden r 2014 Elsevier Inc. All rights reserved.
Glossary Denaturing gradient gel electrophoresis A method for separating deoxyribonucleic acid (DNA) fragments according to their mobility under increasingly denaturing conditions. Necrotizing enterocolitis Bacterial infection in the intestine, primarily of sick or premature newborn infants. Pancreatitis Inflammation of the pancreas. Prebiotics A selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health.
Introduction Probiotics are defined by Food and Agricultural Organization of the United Nations (FAO)/World Health Organization (WHO) as ‘‘live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.’’ One of the most recent definitions of prebiotics is by Gibson et al. (2010) saying that prebiotics are ‘‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health.’’ This definition is still limited to the gastrointestinal tract; however, a possibility to widen it to also include the oral cavity, skin, and urogenital tract is discussed. Fermentation by lactic acid bacteria (LAB), molds, and yeasts is one of the oldest methods for a natural safe preservation of foods. The genus Lactobacillus has played a major role in food preservation. Today most of the probiotic bacterial strains that are used belong to the genus Bifidobacterium and the LAB group, e.g. lactobacilli. These are bacteria that have been safely used in foods by humans for a long time and have acquired a status as ‘generally recognized as safe’ (GRAS). Many prebiotic carbohydrates have their origin in plants and vegetables and occur naturally in chicory root, asparagus, onions, different vegetable roots, wheat, and oats that have been consumed as part of our daily food for thousands of years without any adverse effects. During the past three decades, the number of food products with added pro- and prebiotics has increased considerably. Also new genera, species, and strains are introduced as probiotics. A few cases of adverse effects have been documented in severely
Encyclopedia of Food Safety, Volume 3
Probiotics Live microorganisms, which when administered in adequate amounts, confer a health benefit on the host. Pulsed field gel electrophoresis An electrophoretic technique in which the gel is subjected to electrical fields alternating between different angles, allowing very large DNA fragments to snake through the gel, and hence permitting efficient separation of mixtures of such large fragments. Sepsis A systemic inflammatory response to a infection.
ill patients or immune compromised individuals. This puts the focus on the necessity to handle the safety aspects of pro- and prebiotics in an appropriate way, and especially when probiotics are given to potentially vulnerable persons, the safety issues should be considered with great care. This overview article deals with two kinds of safety issues for probiotics, namely, the potential risk of adverse effects from probiotics and the possibility to improve safety in a product by the use of food-grade bacteria or probiotic bacteria. The article also addresses the risk of adverse effects of prebiotics.
Safety Concerns Linked to the Probiotic Microorganisms General Safety of Probiotics Today both different bacteria and yeasts are used as probiotics (Table 1). Because the most commonly used probiotic bacteria belong to the Lactobacillus and Bifidobacterium genera, most of our knowledge on the general safety of probiotics is based on the experience gained from their use. Despite the common use of probiotics, reported correlations between consumption of probiotics and adverse effects are very rare. Adverse effects have only been reported in seriously ill patients or immune compromised persons, with a very few exceptions. Bifidobacteria and lactobacilli have been isolated from persons with bacteremia; however, no case is known where commercially used probiotic strains have been identified to cause an infection in healthy humans. Furthermore, in Finland, a country where the consumption of probiotics has increased significantly during the
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Table 1 Examples of microorganisms used as probiotics in food, food supplements, and feed Microorganism used as probiotics Genera Lactobacillus Species • Lactobacillus acidophilus • Lactobacillus bulgaricus • Lactobacillus casei • Lactobacillus plantarum • Lactobacillus rhamnosus • Lactobacillus reuteri Genera Bifidobacterium Species • Bifidobacterium bifidum • Bifidobacterium infantis • Bifidobacterium lactis • Bifidobacterium longum Streptococcus Genera Enterococcus • Enterococcus faecalis • Enterococcus faecium Saccharomyces cerevisiae biovar. boulardii Propionibacterium Genera Bacillus • Bacillus subtilis • Bacillus licheniformis Escherichia coli
past decades, no correlation between the increased consumption of probiotics and increase in bacteremia caused by lactobacilli has been identified. The risk of Lactobacillus infection in healthy persons has been calculated by Bernardeau et al. (2006), based on the ingestion and infection frequency of lactobacilli in France, to be approximately one in 10 million individuals during a century of probiotic consumption. This means that lactobacilli are a nearly negligible risk. In a large study supported by National Institutes of Health, 12 databases were searched for studies and reviews on safety of probiotics. It resulted in 622 studies being included in the analyses. The conclusion from this study is that ‘the available evidence in randomized controlled trials does not indicate an increased risk; however, rare adverse events are difficult to assess.’
Safety of Probiotics When Used in Vulnerable Human Populations The cases identified when probiotics have caused illness have been nearly exclusively when they have been given to seriously ill persons, for example, severely immune compromised individuals, people in hospital care, and neonates. Probiotics have been shown to have a potential to significantly lower the risks of infections and the need for antibiotics and thus positively influence the chance to become healthy. However, when used for those purposes in severely ill or hospitalized individuals, a few cases where the probiotic organisms have given an infection have been identified. When probiotics are used under those circumstances, they cannot be considered as food but are used as drugs and therefore a risk–benefit
calculation needs to be done. However today, no probiotics are documented or sold as drugs, but as foods or food supplements, meaning that the risk–benefit calculations are missing. If probiotics are going to be used in the future for these groups at risk, careful assessments of risks must be done.
Areas Where Safety Issues Linked to Probiotics Are Especially Important Transmissible Antibiotic Resistance Humans or animals exposed to probiotic strains carrying antibiotic resistance genes that might be transferred to the commensal microbiota in vivo are at a risk. This issue has been addressed in the European Union (EU) founded project ‘Biosafety Evaluation of Probiotic Lactic Acid Bacteria for Human Consumption’ and in the ‘Assessment and Critical Evaluation of Antibiotic Resistance Transferability in the Food Chain’ project where the antibiotic resistance character of a large number of strains was analyzed and recommendations on the determination of safety was given. The cut-off values for different antibiotics in different bacterial species were set. Antibiotic susceptibility of other strains belonging to the same species can be compared with those cut-off values to judge the possible acquired antibiotic susceptibility in a specific strain. Special focus on the transfer of antibiotic resistance genes and the presence of virulence genes has been given to the genus Enterococcus. The antibiotic resistance questions have also been in focus in the International Organization for Standardization (ISO) and International Dairy Federation joint Action Team on Probiotics.
Probiotics to Severely Ill Individuals In severely ill patients, the gut function is critical and the risk of a breakdown of the gut barrier is increased. This increases the risk of sepsis, systematic inflammation, and multiorgan failure. The potential for probiotics to act beneficially in this group is large; however, the risk of adverse effect is also increased compared with healthy individuals. A number of studies on the incidence of infection and the need for antibiotics in patients undergoing abdominal surgery have shown significantly fewer infections in patients who were given probiotics. In one study, the probiotic prophylaxis in predicted severe acute pancreatitis (PROPATRIA) study, a combination of six strains, belonging to the Lactobacillus and Bifidobacterium genera were given to patients severely ill with pancreatitis. The incidence of infection was similar in the placebo and the probiotic groups; however, the mortality rate was significantly higher in the probiotic group, 16% compared with 6% in the placebo group. The discussions on demands for better risk assessment methods when probiotics are given to the seriously ill patients raised by this study were summarized by Morrow et al. (2012). A large review study by Whelan and Myers (2010) was done, according to Cochrane and Preferred Reporting Item for Systematic Reviews and Meta-Analyses (PRISMA) recommendations, to investigate the possible adverse effects of probiotics given to patients receiving nutritional support. The participants were hospitalized patients – undergoing surgery or transplantation or pancreatitis patients. A total of 53 trials
Safety of Food and Beverages: Safety of Probiotics and Prebiotics
were included where 4131 patients received probiotics and 3643 placebo. According to the outcome of mortality or infection, most trials showed less mortality and less infections or no effect in the probiotic group compared with placebo. There were case reports of adverse effects for 32 patients due to infections with Lactobacillus rhamnosus or Saccharomyces boulardii. The main risk factor was the use of a central venous catheter and increased translocation.
Immune Compromised Persons The gut microbiota is essential for the development and maintenance of the immune function, which also involves maintenance of the gut barrier function. Probiotics might give a great benefit to immune compromised persons; however, this is also a group of individuals where the risk of adverse effects, such as sepsis is large. The mechanisms behind probiotic effects on the immune system are not very well known. Some strains have been seen to stimulate the immune system and enhance the response to pathogens, whereas some other strains can decrease inflammation and allergy. Thus to achieve the desired effects on the immune system, the choice of strains is very important.
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lactobacilli. Acidosis has not been detected in healthy children, but it cannot be ruled out that giving large doses of LAB producing the D(þ)- isomer might be a risk. To avoid this, probiotics given to children shall produce only L()-lactate.
Weight Gain and Probiotics The gut microbiota has been shown to be important for carbohydrate and fat metabolism. The microbiota differs between obese and lean persons and the major difference identified so far is that the diversity is less in obese subjects. These results together with the fact that probiotics have long been used to avoid the need for antibiotics to keep animals raised for meat production healthy, and for quick gain of weight initiated a question on whether the use of probiotics induces obesity. However, it is important to keep in mind that when young animals get probiotics they get fewer infections and grow faster because they are healthy. From this no conclusion on cause of obesity can be drawn. There are studies that have shown that probiotics might influence fat metabolism in a favorable way and reduce fat deposition. Thus today there are no data supporting the view that probiotics contributes to obesity in humans.
Infants The sterile gut of a newborn baby is quickly colonized with different microorganisms, including bifidobacteria, lactobacilli, enterococci, and Escherichia coli, and at 1 year of age the gut microbiota is similar to the adult one. The colonization of the gut is crucial for its maturation and for the development and maintenance of the immune system. Many studies have been conducted on probiotics in healthy fullterm infants with positive effects on health and no adverse effects. Still, long-term effects are not well studied and methodologies to identify infants at possible risk for adverse effects are missing. Thus there is a need for more studies on longterm effects of probiotics in term infants. Preterm infants are often separated from their mothers and raised during their first period of life under strict hygienic conditions, with impaired enteral feeding and exposure to antibiotics. This means that the colonization of the gut is very different from that of term infants, for example, fewer species are colonizing the gut and the colonization with bifidobacteria is delayed. A risk in preterm infants is the development of necrotizing enterocolitis (NEC), which is a rare but severe consequence of an immature gut and impaired mucosal barrier. Intervention studies with probiotic strains of lactobacilli, bifidobacteria, or streptococci have been seen to reduce the incidence and severity of NEC. Neither in this study by Dani et al. (2002) nor in a study by Lin et al. (2008), altogether including 729 infants, was sepsis identified. Thus in preterm infants, the use of probiotics to diminish the risk of NEC is very promising but risk–benefit estimation needs to be done. As in term infants the long-term effects of probiotics is not known and need to be investigated. Humans produce L()-lactate only and all the D(þ)-lactate present in humans is produced by microorganisms or through microbial conversion of L()-lactate to D(þ)-lactate. If large amounts of D-lactate is built up in children, it may create acidosis. This has been detected in children with short bowel syndrome and with a gut microbiota dominated by
Safety Regulations and Probiotics In Europe, the European Food Safety Authority (EFSA) has introduced an approach for safety assessment of bacterial strains including probiotics called ‘qualified presumption of safety’ (QPS). The approach was introduced in 2007 and is updated every year for new microorganisms. The safety assessment of specific taxonomic groups of microorganisms is done according to taxonomic identity of the strain, body of knowledge, possible safety concern (pathogenicity), and intended end use. If the taxonomic identity does not raise safety concerns, the taxonomic unit can be put on the QPS list and the only further safety studies needed are on antibiotic resistance. If a microorganism does not belong on the QPS list, considerable assessments of food safety are needed before it can be used in foods. The genera Lactobacillus and Bifidobacterium are all on the QPS list. FAO/WHO introduced guidelines for the use of probiotics in foods. This includes establishing the safe use in traditional products, absence of transmissible antibiotic resistance, absence of risks with virulence properties, no production of D-lactate, no toxin production, no hemolytic properties, no side effects in human interventions, and epidemiologically known to be safe (the latter is done postlaunching). No proper instructions on how the documentation shall be done are available. Lactobacilli, bifidobacteria, and Saccharomyces cerevisiae belong to the organisms stated as GRAS. Canada has recently developed guidelines for the industry called ‘recommendations for the evidence requirements for efficacy, safety, and quality of natural health products containing probiotics.’ In Norway, the Norwegian Scientific Committee for Food Safety has evaluated the safety of a few probiotic strains. Guidelines for the regulation of probiotics in food are to be introduced. In China, guidelines are available since 2005 and all foods with a health claim require an application to and approval by
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the State Food and Drug Administration concerning efficacy, safety, and quality.
Basic Safety Documentation for Probiotics Based on the regulations in different countries and from the available literature, the following issues are important for a proper safety assessment of a probiotic organism:
• • • • • • • • •
Proper taxonomic identification at strain level. The nature of the microbe (linked to taxonomy). Absence of transmissible antibiotic resistance genes. Genetic stability. Absence of infectivity and translocation properties. Absence of toxin production. The population for which the product is intended for must be considered and if it is intended for any vulnerable population, extended risk assessments need to be done. Method of administration. For production of the probiotic products, good manufacturing procedures need to be used to ensure the hygiene and safety standards needed.
Sequencing of the genome of a probiotic organism is a useful tool to analyze the presence of possible genes that are linked to unwanted characteristics, such as antibiotic resistance and toxin production. Modern molecular biological methods should be used for proper taxonomic identification at species and strain levels. These include the analysis of the ribosomal ribonucleic acid subunit 16S, the use of strain-specific primers, and deoxyribonucleic acid finger printing techniques, such as ribotyping, pulsed field gel electrophoresis, and denaturing gradient gel electrophoresis.
Safety Issues Linked to Prebiotics The target microbiota of prebiotics is dominated by the genera Lactobacillus and Bifidobacterium. It is the bifidobacteria that have been in the main focus and the possibility of these influencing the gut microbiota and increasing the number of bifidobacteria has gained much attention. The requirement for a prebiotic was more precisely described by Gibson et al. as a substance resistant to gastric acidity and hydrolyzes by mammalian enzymes and adsorption. It can be fermented by intestinal microflora and it selectively stimulates the growth and/or activity of intestinal bacteria associated with health and wellbeing. Many prebiotic carbohydrates such as inulin and fructooligosaccharides (FOS) are natural constituents of foods. In the US, inulin and FOS have a status as GRAS since 1999. No severe side effects have been seen with prebiotics and the major side effect attributed to prebiotics is cause of osmotic diarrhea, rumbling, and abdominal pain due to bloating, cramping, and/or flatulence. This is because the polysaccharides and oligosaccharides are not broken down by intestinal enzymes but transported to the colon where they are fermented. Daily high doses, 40–50 g, have been seen to cause osmotic effect, whereas low doses 2.5–10 g may cause
Table 2 Examples of carbohydrate used as prebiotics in food and feed supplements Carbohydrates with documented prebiotic effect Fructans • Short chain fructooligosaccharides • Fructooligosaccharides • Inulin Galactooligosaccharides • Transgalactooligosaccharides • Lactulose Carbohydrates suggested to be prebiotics Polydextrose Glucans Resistant starch Pectins
fermentation and possible flatulence. It appears that the chain length of inulin-type prebiotics influence the severity of side effect, that is, shorter chain length increases the side effect. The theory behind this is that shorter inulin molecules are metabolized primarily in the proximal colon and thus are more rapidly fermented, whereas longer chains are fermented in the distal colon. The daily dose required to achieve a positive effect on the gut microbiota or on other health effects from prebiotics is in most studies 2.5–10 g and most products on the market today have doses of 1.5–5 g per portion. This means that the dose for a positive effect touches the dose for a mild side effect. Most probably the large majority of humans can eat this amount without side effects but if more and more products are being fortified with prebiotics, this needs additional attention (Table 2).
Discrimination between the EFSA Health Claim Regulation and Safety Issues When EU launched the Regulation (EC) No 1924/2006 in 2007 for health claims, the basis for the regulation was a concept of consumer protection, that is, the labeling of foods should be appropriate according to nutrients and health claims. So far no health claim for pro- or prebiotics has been approved. Van Loveren et al. (2012) have summarized the reason for health claim rejection and link to safety. The reason for health claim rejection is not a matter of safety of probiotic or prebiotics. The reason is instead linked to that in some cases the probiotic bacteria has not been taxonomically identified to the appropriate detailed level and before the regulations, studies on pro- and prebiotics were done more in an explorative way and not according to the current claim regulations. In 2011 and 2012 EFSA published guidelines for human intervention studies in different areas, and the application of those guidelines will facilitate studies and dossiers for approval of health claims. If a probiotic bacterium is included on the EFSA QPS list, which also most probiotics are, safety is not an issue for getting health claims. Thus the common demands for health claims and for safety judgment is that the probiotic or prebiotic used is appropriately
Safety of Food and Beverages: Safety of Probiotics and Prebiotics
identified. However, if the probiotic strain is not on the QPS list, safety considerations are recommended according to the novel food regulations.
Use of Probiotic Bacteria/LAB to Increase the Safety of a Product Use of Microorganisms for Natural Biopreservation of Foods LAB, fungi, and molds have been used for the preservation of foods for thousands of years. The most common method of biopreservation is fermentation. The preservation effect is due to the production of organic acid, diacetyl, and hydrogen peroxide but also to specific antimicrobial compounds such as bacteriocins and antifungal peptides. Mathematical models and predictive microbiology have been used to address the importance of process factors together with acid and bacteriocin production. When the food industry now is looking for natural preservation methods, the use of LAB with its GRAS status is attractive. Bacteriocin-producing LAB have been applied to improve food quality and safety in sourdough and sausages production and in cheese production for competition, antilisteria, and anticlostridia purposes. The most studied bacteriocin is nisin for its preservative effects in foods, especially cheese. Also the potential of application within the malting and brewing process to diminish the growth of naturally occurring unwanted microorganisms has been suggested. The possibility to use bacteriocin-producing bacteria as probiotics for human and animal health to lower the risk of infection and for therapeutic purposes is an interesting possibility with a potential to lower the wide use of antibiotics. Bacteriocin production might also strengthen the competiveness of a probiotic bacteria in complex microbial communities such as those in the gut.
Binding of Toxic Compounds (Mycotoxins, Heterocyclic Aromatic Amines, and Heavy Metals) Production of mycotoxins in foods and feeds create severe spoilage and is a threat to human health. The mycotoxins might be carcinogenic, immunotoxic, neurotoxic, nephrotoxic, or hepatotoxic. The use of LAB as natural preservatives to produce organic acids and bacteriocins to diminish the growth of the unwanted microflora as well as the capacity of LAB to bind mycotoxins is an interesting natural preservation method. One of the most common mycotoxin is aflatoxin, which is produced by Aspergillus strains of fungi. The ability to bind aflatoxin is strain dependent, and binding of toxic substances has been seen both in in vitro and in vivo situations. Different examples on the potential for probiotics to bind and detoxify mycotoxins have been described by Amalaradjou and Buhnia (2012) and Salminen et al. (2010), for example, different probiotic strains have been seen to bind and reduce the bioavailability of different forms of aflatoxin, including the most potent one aflatoxin B1, ochratoxin A, fusarium toxins, and patulin. Studies have shown that the mechanism behind removing of mycotoxins most probably is binding of the toxins and not a metabolic process since, for example, both dead and live probiotics have the ability to remove the toxins.
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Similarly, strains of L. rhamnosus, Propionibacterium, and S. cerevisiae have been shown to bind aflatoxin B1 also in the chicken or ruminant gastrointestinal tract and thus have the potential to be used as a feed additive to reduce the bioavailability of aflatoxin. Only a few studies have been performed in humans and a study in China showed that feeding a dietary supplement with specific strains of probiotics reduces the urinary excretion of aflatoxin B1. It should be noted that the binding capacity seems to be strain specific. The production of toxins by cyanobacteria in aquatic and marine environments and specifically in drinking water is a risk of poisoning. The possibility to remove microcystin toxins produced by cyanobacteria in drinking water has been investigated using various lactobacilli and bifidobacteria separately or in combination with promising results. When meat is cooked at 150–300 1C heterocyclic aromatic amines (HAA) are formed. These compounds have a high mutagenic potential and might contribute to gastrointestinal cancer. The possibility to utilize probiotic bacteria to eliminate theses mutagenic substances is interesting and investigations by Nowak and Libudzisz have shown in vitro that the probiotic bacteria Lactobacillus casei DN-114001 reduces the amount of three different HAA compounds by 27–99%, indicating that the toxins are metabolized. The use of different microorganisms to bind metal cations has been studied for waste management. This could be expanded to foods by the use of food-grade microorganisms. The potential to use probiotic bacteria in this area for the decontamination of foods and the intestine from heavy metals have been investigated and the ability to bind, for example, lead and cadmium, has been shown to be dependent on the strain and pH.
See also: Public Health Measures: Assessment of Novel Foods and Ingredients. Safety of Food and Beverages: Probiotics and Prebiotics; Safety Consideration in Developing Functional Foods
References Bernardeau M, Guguen M, and Vernoux JP (2006) Beneficial lactobacilli in food and feed: Long-term use, biodiversity and proposal for specific and realistic safety assessment. FEMS Microbiology Reviews 30: 487–513. Dani C, Biadaioli R, Bertini G, Martelli E, and Rubaltelli FF (2002) Probiotic feeding in prevention of urinary tract infection, bacterial sepsis and necrotizing enterocolitis in preterm infants. A prospective double-blind study. Biology of the Neonate 82: 103–108. Gibson GR, Scott KP, Rastall RA, et al. (2010) Dietary prebiotics: Current status and definition. Food Science and Technology Bulletin: Functional Foods 7: 1–19. Lin HC, Hsu CH, Chen HL, et al. (2008) Oral probiotics prevents necrotizing enterocolitis in very low birth weight preterm infants: A multicenter, randomized, controlled trial. Pediatrics 122: 693–700. van Loveren H, Sanz Y, and Salminen S (2012) Health claims in Europe: Probiotics and prebiotics as case examples. Annual Review of Food Science and Technology 3: 247–261. Morrow LE, Gogineni V, and Malesker MA (2012) Probiotic, prebiotic, and symbiotic use in critically ill patients. Current Opinion in Critical Care 18: 186–191. Salminen S, Nybom S, Meriluoto J, Collado MC, Vesterlund S, and El-Nezami H (2010) Interaction of probiotics and pathogens – benefit to human health. Current Opinion in Biotechnology 21: 157–167.
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Further Reading De Vuyst L and Leroy F (2007) Bacteriocins from lactic acid bacteria: Production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13: 194–199. FAO/WHO (2006) Probiotics in foods. Health and nutritional properties and guidelines for evaluation. FAO Food and Nutrition Paper 85. Rome: FAO/WHO. Available at: ftp://ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf (accessed on 8 May 2013). van Hoek AHAM, Margolles A, Doming KJ, et al. (2008) Molecular assessment of erythromycin and tetracycline resistance genes in lactic acid bacteria and bifidobacteria and their relation to the phenotypic resistance. International Journal of Probiotics and Prebiotics 3: 271–280. Lahtinen SJ, Davis E, and Ouwehand AC (2012) Lactobacillus species causing obesity in humans: Where is the evidence? Beneficial Microbes 3: 171–174. Nowak A and Libudzisz Z (2009) Ability of probiotic Lactobacillus casei DN 114001 to bind or/and metabolize heterocyclic aromatic amines in vitro. European Journal of Nutrition 48: 419–427. Sanders ME, Akkermans LMA, Haller D, et al. (2010) Safety assessment of probiotics for human use. Gut Microbes 1(3): 164–185. Vankerckhoven V, Huys G, Vancanneyt M, et al. (2008) Biosafety assessment of probiotics used for human consumption: Recommendations from the EUPROSAFE project. Trends in Food Science and Technology 19: 102–114.
Whelan K and Myers CE (2010) Safety of probiotics in patients receiving nutritional support: A systematic review of case reports, randomized controlled trials, and nonrandomized trials. American Journal of Clinical Nutrition 91: 687–703.
Relevant Websites www.ahrq.gov/clinic/tp/probiotictp.htm Agency for Healthcare Research and Quality: Safety of Probiotics to Reduce Risk and Prevent or Treat Disease (2011) Evidence report/Technology assessment No. 200. AHRQ Publication No.11-E007. www.efsa.europa.eu/en/topics/topic/qps.htm EFSA. ftp://ftp.fao.org/es/esn/food/wgreport2.pdf FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food (2002). http://www.SFDA.com State Food and Drug Administration (SFDA) in China.
Safety of Probiotics and Prebiotics UK Svensson, UKS Life Science Consulting AB, Lund, Sweden; Lund University, Lund, Sweden J Ha˚kansson, ASIC, Arla Foods, Stockholm, Sweden r 2014 Elsevier Inc. All rights reserved.
Glossary Denaturing gradient gel electrophoresis A method for separating deoxyribonucleic acid (DNA) fragments according to their mobility under increasingly denaturing conditions. Necrotizing enterocolitis Bacterial infection in the intestine, primarily of sick or premature newborn infants. Pancreatitis Inflammation of the pancreas. Prebiotics A selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health.
Introduction Probiotics are defined by Food and Agricultural Organization of the United Nations (FAO)/World Health Organization (WHO) as ‘‘live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.’’ One of the most recent definitions of prebiotics is by Gibson et al. (2010) saying that prebiotics are ‘‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health.’’ This definition is still limited to the gastrointestinal tract; however, a possibility to widen it to also include the oral cavity, skin, and urogenital tract is discussed. Fermentation by lactic acid bacteria (LAB), molds, and yeasts is one of the oldest methods for a natural safe preservation of foods. The genus Lactobacillus has played a major role in food preservation. Today most of the probiotic bacterial strains that are used belong to the genus Bifidobacterium and the LAB group, e.g. lactobacilli. These are bacteria that have been safely used in foods by humans for a long time and have acquired a status as ‘generally recognized as safe’ (GRAS). Many prebiotic carbohydrates have their origin in plants and vegetables and occur naturally in chicory root, asparagus, onions, different vegetable roots, wheat, and oats that have been consumed as part of our daily food for thousands of years without any adverse effects. During the past three decades, the number of food products with added pro- and prebiotics has increased considerably. Also new genera, species, and strains are introduced as probiotics. A few cases of adverse effects have been documented in severely
Encyclopedia of Food Safety, Volume 3
Probiotics Live microorganisms, which when administered in adequate amounts, confer a health benefit on the host. Pulsed field gel electrophoresis An electrophoretic technique in which the gel is subjected to electrical fields alternating between different angles, allowing very large DNA fragments to snake through the gel, and hence permitting efficient separation of mixtures of such large fragments. Sepsis A systemic inflammatory response to a infection.
ill patients or immune compromised individuals. This puts the focus on the necessity to handle the safety aspects of pro- and prebiotics in an appropriate way, and especially when probiotics are given to potentially vulnerable persons, the safety issues should be considered with great care. This overview article deals with two kinds of safety issues for probiotics, namely, the potential risk of adverse effects from probiotics and the possibility to improve safety in a product by the use of food-grade bacteria or probiotic bacteria. The article also addresses the risk of adverse effects of prebiotics.
Safety Concerns Linked to the Probiotic Microorganisms General Safety of Probiotics Today both different bacteria and yeasts are used as probiotics (Table 1). Because the most commonly used probiotic bacteria belong to the Lactobacillus and Bifidobacterium genera, most of our knowledge on the general safety of probiotics is based on the experience gained from their use. Despite the common use of probiotics, reported correlations between consumption of probiotics and adverse effects are very rare. Adverse effects have only been reported in seriously ill patients or immune compromised persons, with a very few exceptions. Bifidobacteria and lactobacilli have been isolated from persons with bacteremia; however, no case is known where commercially used probiotic strains have been identified to cause an infection in healthy humans. Furthermore, in Finland, a country where the consumption of probiotics has increased significantly during the
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Table 1 Examples of microorganisms used as probiotics in food, food supplements, and feed Microorganism used as probiotics Genera Lactobacillus Species • Lactobacillus acidophilus • Lactobacillus bulgaricus • Lactobacillus casei • Lactobacillus plantarum • Lactobacillus rhamnosus • Lactobacillus reuteri Genera Bifidobacterium Species • Bifidobacterium bifidum • Bifidobacterium infantis • Bifidobacterium lactis • Bifidobacterium longum Streptococcus Genera Enterococcus • Enterococcus faecalis • Enterococcus faecium Saccharomyces cerevisiae biovar. boulardii Propionibacterium Genera Bacillus • Bacillus subtilis • Bacillus licheniformis Escherichia coli
past decades, no correlation between the increased consumption of probiotics and increase in bacteremia caused by lactobacilli has been identified. The risk of Lactobacillus infection in healthy persons has been calculated by Bernardeau et al. (2006), based on the ingestion and infection frequency of lactobacilli in France, to be approximately one in 10 million individuals during a century of probiotic consumption. This means that lactobacilli are a nearly negligible risk. In a large study supported by National Institutes of Health, 12 databases were searched for studies and reviews on safety of probiotics. It resulted in 622 studies being included in the analyses. The conclusion from this study is that ‘the available evidence in randomized controlled trials does not indicate an increased risk; however, rare adverse events are difficult to assess.’
Safety of Probiotics When Used in Vulnerable Human Populations The cases identified when probiotics have caused illness have been nearly exclusively when they have been given to seriously ill persons, for example, severely immune compromised individuals, people in hospital care, and neonates. Probiotics have been shown to have a potential to significantly lower the risks of infections and the need for antibiotics and thus positively influence the chance to become healthy. However, when used for those purposes in severely ill or hospitalized individuals, a few cases where the probiotic organisms have given an infection have been identified. When probiotics are used under those circumstances, they cannot be considered as food but are used as drugs and therefore a risk–benefit
calculation needs to be done. However today, no probiotics are documented or sold as drugs, but as foods or food supplements, meaning that the risk–benefit calculations are missing. If probiotics are going to be used in the future for these groups at risk, careful assessments of risks must be done.
Areas Where Safety Issues Linked to Probiotics Are Especially Important Transmissible Antibiotic Resistance Humans or animals exposed to probiotic strains carrying antibiotic resistance genes that might be transferred to the commensal microbiota in vivo are at a risk. This issue has been addressed in the European Union (EU) founded project ‘Biosafety Evaluation of Probiotic Lactic Acid Bacteria for Human Consumption’ and in the ‘Assessment and Critical Evaluation of Antibiotic Resistance Transferability in the Food Chain’ project where the antibiotic resistance character of a large number of strains was analyzed and recommendations on the determination of safety was given. The cut-off values for different antibiotics in different bacterial species were set. Antibiotic susceptibility of other strains belonging to the same species can be compared with those cut-off values to judge the possible acquired antibiotic susceptibility in a specific strain. Special focus on the transfer of antibiotic resistance genes and the presence of virulence genes has been given to the genus Enterococcus. The antibiotic resistance questions have also been in focus in the International Organization for Standardization (ISO) and International Dairy Federation joint Action Team on Probiotics.
Probiotics to Severely Ill Individuals In severely ill patients, the gut function is critical and the risk of a breakdown of the gut barrier is increased. This increases the risk of sepsis, systematic inflammation, and multiorgan failure. The potential for probiotics to act beneficially in this group is large; however, the risk of adverse effect is also increased compared with healthy individuals. A number of studies on the incidence of infection and the need for antibiotics in patients undergoing abdominal surgery have shown significantly fewer infections in patients who were given probiotics. In one study, the probiotic prophylaxis in predicted severe acute pancreatitis (PROPATRIA) study, a combination of six strains, belonging to the Lactobacillus and Bifidobacterium genera were given to patients severely ill with pancreatitis. The incidence of infection was similar in the placebo and the probiotic groups; however, the mortality rate was significantly higher in the probiotic group, 16% compared with 6% in the placebo group. The discussions on demands for better risk assessment methods when probiotics are given to the seriously ill patients raised by this study were summarized by Morrow et al. (2012). A large review study by Whelan and Myers (2010) was done, according to Cochrane and Preferred Reporting Item for Systematic Reviews and Meta-Analyses (PRISMA) recommendations, to investigate the possible adverse effects of probiotics given to patients receiving nutritional support. The participants were hospitalized patients – undergoing surgery or transplantation or pancreatitis patients. A total of 53 trials
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were included where 4131 patients received probiotics and 3643 placebo. According to the outcome of mortality or infection, most trials showed less mortality and less infections or no effect in the probiotic group compared with placebo. There were case reports of adverse effects for 32 patients due to infections with Lactobacillus rhamnosus or Saccharomyces boulardii. The main risk factor was the use of a central venous catheter and increased translocation.
Immune Compromised Persons The gut microbiota is essential for the development and maintenance of the immune function, which also involves maintenance of the gut barrier function. Probiotics might give a great benefit to immune compromised persons; however, this is also a group of individuals where the risk of adverse effects, such as sepsis is large. The mechanisms behind probiotic effects on the immune system are not very well known. Some strains have been seen to stimulate the immune system and enhance the response to pathogens, whereas some other strains can decrease inflammation and allergy. Thus to achieve the desired effects on the immune system, the choice of strains is very important.
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lactobacilli. Acidosis has not been detected in healthy children, but it cannot be ruled out that giving large doses of LAB producing the D(þ)- isomer might be a risk. To avoid this, probiotics given to children shall produce only L()-lactate.
Weight Gain and Probiotics The gut microbiota has been shown to be important for carbohydrate and fat metabolism. The microbiota differs between obese and lean persons and the major difference identified so far is that the diversity is less in obese subjects. These results together with the fact that probiotics have long been used to avoid the need for antibiotics to keep animals raised for meat production healthy, and for quick gain of weight initiated a question on whether the use of probiotics induces obesity. However, it is important to keep in mind that when young animals get probiotics they get fewer infections and grow faster because they are healthy. From this no conclusion on cause of obesity can be drawn. There are studies that have shown that probiotics might influence fat metabolism in a favorable way and reduce fat deposition. Thus today there are no data supporting the view that probiotics contributes to obesity in humans.
Infants The sterile gut of a newborn baby is quickly colonized with different microorganisms, including bifidobacteria, lactobacilli, enterococci, and Escherichia coli, and at 1 year of age the gut microbiota is similar to the adult one. The colonization of the gut is crucial for its maturation and for the development and maintenance of the immune system. Many studies have been conducted on probiotics in healthy fullterm infants with positive effects on health and no adverse effects. Still, long-term effects are not well studied and methodologies to identify infants at possible risk for adverse effects are missing. Thus there is a need for more studies on longterm effects of probiotics in term infants. Preterm infants are often separated from their mothers and raised during their first period of life under strict hygienic conditions, with impaired enteral feeding and exposure to antibiotics. This means that the colonization of the gut is very different from that of term infants, for example, fewer species are colonizing the gut and the colonization with bifidobacteria is delayed. A risk in preterm infants is the development of necrotizing enterocolitis (NEC), which is a rare but severe consequence of an immature gut and impaired mucosal barrier. Intervention studies with probiotic strains of lactobacilli, bifidobacteria, or streptococci have been seen to reduce the incidence and severity of NEC. Neither in this study by Dani et al. (2002) nor in a study by Lin et al. (2008), altogether including 729 infants, was sepsis identified. Thus in preterm infants, the use of probiotics to diminish the risk of NEC is very promising but risk–benefit estimation needs to be done. As in term infants the long-term effects of probiotics is not known and need to be investigated. Humans produce L()-lactate only and all the D(þ)-lactate present in humans is produced by microorganisms or through microbial conversion of L()-lactate to D(þ)-lactate. If large amounts of D-lactate is built up in children, it may create acidosis. This has been detected in children with short bowel syndrome and with a gut microbiota dominated by
Safety Regulations and Probiotics In Europe, the European Food Safety Authority (EFSA) has introduced an approach for safety assessment of bacterial strains including probiotics called ‘qualified presumption of safety’ (QPS). The approach was introduced in 2007 and is updated every year for new microorganisms. The safety assessment of specific taxonomic groups of microorganisms is done according to taxonomic identity of the strain, body of knowledge, possible safety concern (pathogenicity), and intended end use. If the taxonomic identity does not raise safety concerns, the taxonomic unit can be put on the QPS list and the only further safety studies needed are on antibiotic resistance. If a microorganism does not belong on the QPS list, considerable assessments of food safety are needed before it can be used in foods. The genera Lactobacillus and Bifidobacterium are all on the QPS list. FAO/WHO introduced guidelines for the use of probiotics in foods. This includes establishing the safe use in traditional products, absence of transmissible antibiotic resistance, absence of risks with virulence properties, no production of D-lactate, no toxin production, no hemolytic properties, no side effects in human interventions, and epidemiologically known to be safe (the latter is done postlaunching). No proper instructions on how the documentation shall be done are available. Lactobacilli, bifidobacteria, and Saccharomyces cerevisiae belong to the organisms stated as GRAS. Canada has recently developed guidelines for the industry called ‘recommendations for the evidence requirements for efficacy, safety, and quality of natural health products containing probiotics.’ In Norway, the Norwegian Scientific Committee for Food Safety has evaluated the safety of a few probiotic strains. Guidelines for the regulation of probiotics in food are to be introduced. In China, guidelines are available since 2005 and all foods with a health claim require an application to and approval by
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the State Food and Drug Administration concerning efficacy, safety, and quality.
Basic Safety Documentation for Probiotics Based on the regulations in different countries and from the available literature, the following issues are important for a proper safety assessment of a probiotic organism:
• • • • • • • • •
Proper taxonomic identification at strain level. The nature of the microbe (linked to taxonomy). Absence of transmissible antibiotic resistance genes. Genetic stability. Absence of infectivity and translocation properties. Absence of toxin production. The population for which the product is intended for must be considered and if it is intended for any vulnerable population, extended risk assessments need to be done. Method of administration. For production of the probiotic products, good manufacturing procedures need to be used to ensure the hygiene and safety standards needed.
Sequencing of the genome of a probiotic organism is a useful tool to analyze the presence of possible genes that are linked to unwanted characteristics, such as antibiotic resistance and toxin production. Modern molecular biological methods should be used for proper taxonomic identification at species and strain levels. These include the analysis of the ribosomal ribonucleic acid subunit 16S, the use of strain-specific primers, and deoxyribonucleic acid finger printing techniques, such as ribotyping, pulsed field gel electrophoresis, and denaturing gradient gel electrophoresis.
Safety Issues Linked to Prebiotics The target microbiota of prebiotics is dominated by the genera Lactobacillus and Bifidobacterium. It is the bifidobacteria that have been in the main focus and the possibility of these influencing the gut microbiota and increasing the number of bifidobacteria has gained much attention. The requirement for a prebiotic was more precisely described by Gibson et al. as a substance resistant to gastric acidity and hydrolyzes by mammalian enzymes and adsorption. It can be fermented by intestinal microflora and it selectively stimulates the growth and/or activity of intestinal bacteria associated with health and wellbeing. Many prebiotic carbohydrates such as inulin and fructooligosaccharides (FOS) are natural constituents of foods. In the US, inulin and FOS have a status as GRAS since 1999. No severe side effects have been seen with prebiotics and the major side effect attributed to prebiotics is cause of osmotic diarrhea, rumbling, and abdominal pain due to bloating, cramping, and/or flatulence. This is because the polysaccharides and oligosaccharides are not broken down by intestinal enzymes but transported to the colon where they are fermented. Daily high doses, 40–50 g, have been seen to cause osmotic effect, whereas low doses 2.5–10 g may cause
Table 2 Examples of carbohydrate used as prebiotics in food and feed supplements Carbohydrates with documented prebiotic effect Fructans • Short chain fructooligosaccharides • Fructooligosaccharides • Inulin Galactooligosaccharides • Transgalactooligosaccharides • Lactulose Carbohydrates suggested to be prebiotics Polydextrose Glucans Resistant starch Pectins
fermentation and possible flatulence. It appears that the chain length of inulin-type prebiotics influence the severity of side effect, that is, shorter chain length increases the side effect. The theory behind this is that shorter inulin molecules are metabolized primarily in the proximal colon and thus are more rapidly fermented, whereas longer chains are fermented in the distal colon. The daily dose required to achieve a positive effect on the gut microbiota or on other health effects from prebiotics is in most studies 2.5–10 g and most products on the market today have doses of 1.5–5 g per portion. This means that the dose for a positive effect touches the dose for a mild side effect. Most probably the large majority of humans can eat this amount without side effects but if more and more products are being fortified with prebiotics, this needs additional attention (Table 2).
Discrimination between the EFSA Health Claim Regulation and Safety Issues When EU launched the Regulation (EC) No 1924/2006 in 2007 for health claims, the basis for the regulation was a concept of consumer protection, that is, the labeling of foods should be appropriate according to nutrients and health claims. So far no health claim for pro- or prebiotics has been approved. Van Loveren et al. (2012) have summarized the reason for health claim rejection and link to safety. The reason for health claim rejection is not a matter of safety of probiotic or prebiotics. The reason is instead linked to that in some cases the probiotic bacteria has not been taxonomically identified to the appropriate detailed level and before the regulations, studies on pro- and prebiotics were done more in an explorative way and not according to the current claim regulations. In 2011 and 2012 EFSA published guidelines for human intervention studies in different areas, and the application of those guidelines will facilitate studies and dossiers for approval of health claims. If a probiotic bacterium is included on the EFSA QPS list, which also most probiotics are, safety is not an issue for getting health claims. Thus the common demands for health claims and for safety judgment is that the probiotic or prebiotic used is appropriately
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identified. However, if the probiotic strain is not on the QPS list, safety considerations are recommended according to the novel food regulations.
Use of Probiotic Bacteria/LAB to Increase the Safety of a Product Use of Microorganisms for Natural Biopreservation of Foods LAB, fungi, and molds have been used for the preservation of foods for thousands of years. The most common method of biopreservation is fermentation. The preservation effect is due to the production of organic acid, diacetyl, and hydrogen peroxide but also to specific antimicrobial compounds such as bacteriocins and antifungal peptides. Mathematical models and predictive microbiology have been used to address the importance of process factors together with acid and bacteriocin production. When the food industry now is looking for natural preservation methods, the use of LAB with its GRAS status is attractive. Bacteriocin-producing LAB have been applied to improve food quality and safety in sourdough and sausages production and in cheese production for competition, antilisteria, and anticlostridia purposes. The most studied bacteriocin is nisin for its preservative effects in foods, especially cheese. Also the potential of application within the malting and brewing process to diminish the growth of naturally occurring unwanted microorganisms has been suggested. The possibility to use bacteriocin-producing bacteria as probiotics for human and animal health to lower the risk of infection and for therapeutic purposes is an interesting possibility with a potential to lower the wide use of antibiotics. Bacteriocin production might also strengthen the competiveness of a probiotic bacteria in complex microbial communities such as those in the gut.
Binding of Toxic Compounds (Mycotoxins, Heterocyclic Aromatic Amines, and Heavy Metals) Production of mycotoxins in foods and feeds create severe spoilage and is a threat to human health. The mycotoxins might be carcinogenic, immunotoxic, neurotoxic, nephrotoxic, or hepatotoxic. The use of LAB as natural preservatives to produce organic acids and bacteriocins to diminish the growth of the unwanted microflora as well as the capacity of LAB to bind mycotoxins is an interesting natural preservation method. One of the most common mycotoxin is aflatoxin, which is produced by Aspergillus strains of fungi. The ability to bind aflatoxin is strain dependent, and binding of toxic substances has been seen both in in vitro and in vivo situations. Different examples on the potential for probiotics to bind and detoxify mycotoxins have been described by Amalaradjou and Buhnia (2012) and Salminen et al. (2010), for example, different probiotic strains have been seen to bind and reduce the bioavailability of different forms of aflatoxin, including the most potent one aflatoxin B1, ochratoxin A, fusarium toxins, and patulin. Studies have shown that the mechanism behind removing of mycotoxins most probably is binding of the toxins and not a metabolic process since, for example, both dead and live probiotics have the ability to remove the toxins.
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Similarly, strains of L. rhamnosus, Propionibacterium, and S. cerevisiae have been shown to bind aflatoxin B1 also in the chicken or ruminant gastrointestinal tract and thus have the potential to be used as a feed additive to reduce the bioavailability of aflatoxin. Only a few studies have been performed in humans and a study in China showed that feeding a dietary supplement with specific strains of probiotics reduces the urinary excretion of aflatoxin B1. It should be noted that the binding capacity seems to be strain specific. The production of toxins by cyanobacteria in aquatic and marine environments and specifically in drinking water is a risk of poisoning. The possibility to remove microcystin toxins produced by cyanobacteria in drinking water has been investigated using various lactobacilli and bifidobacteria separately or in combination with promising results. When meat is cooked at 150–300 1C heterocyclic aromatic amines (HAA) are formed. These compounds have a high mutagenic potential and might contribute to gastrointestinal cancer. The possibility to utilize probiotic bacteria to eliminate theses mutagenic substances is interesting and investigations by Nowak and Libudzisz have shown in vitro that the probiotic bacteria Lactobacillus casei DN-114001 reduces the amount of three different HAA compounds by 27–99%, indicating that the toxins are metabolized. The use of different microorganisms to bind metal cations has been studied for waste management. This could be expanded to foods by the use of food-grade microorganisms. The potential to use probiotic bacteria in this area for the decontamination of foods and the intestine from heavy metals have been investigated and the ability to bind, for example, lead and cadmium, has been shown to be dependent on the strain and pH.
See also: Public Health Measures: Assessment of Novel Foods and Ingredients. Safety of Food and Beverages: Probiotics and Prebiotics; Safety Consideration in Developing Functional Foods
References Bernardeau M, Guguen M, and Vernoux JP (2006) Beneficial lactobacilli in food and feed: Long-term use, biodiversity and proposal for specific and realistic safety assessment. FEMS Microbiology Reviews 30: 487–513. Dani C, Biadaioli R, Bertini G, Martelli E, and Rubaltelli FF (2002) Probiotic feeding in prevention of urinary tract infection, bacterial sepsis and necrotizing enterocolitis in preterm infants. A prospective double-blind study. Biology of the Neonate 82: 103–108. Gibson GR, Scott KP, Rastall RA, et al. (2010) Dietary prebiotics: Current status and definition. Food Science and Technology Bulletin: Functional Foods 7: 1–19. Lin HC, Hsu CH, Chen HL, et al. (2008) Oral probiotics prevents necrotizing enterocolitis in very low birth weight preterm infants: A multicenter, randomized, controlled trial. Pediatrics 122: 693–700. van Loveren H, Sanz Y, and Salminen S (2012) Health claims in Europe: Probiotics and prebiotics as case examples. Annual Review of Food Science and Technology 3: 247–261. Morrow LE, Gogineni V, and Malesker MA (2012) Probiotic, prebiotic, and symbiotic use in critically ill patients. Current Opinion in Critical Care 18: 186–191. Salminen S, Nybom S, Meriluoto J, Collado MC, Vesterlund S, and El-Nezami H (2010) Interaction of probiotics and pathogens – benefit to human health. Current Opinion in Biotechnology 21: 157–167.
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Further Reading De Vuyst L and Leroy F (2007) Bacteriocins from lactic acid bacteria: Production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13: 194–199. FAO/WHO (2006) Probiotics in foods. Health and nutritional properties and guidelines for evaluation. FAO Food and Nutrition Paper 85. Rome: FAO/WHO. Available at: ftp://ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf (accessed on 8 May 2013). van Hoek AHAM, Margolles A, Doming KJ, et al. (2008) Molecular assessment of erythromycin and tetracycline resistance genes in lactic acid bacteria and bifidobacteria and their relation to the phenotypic resistance. International Journal of Probiotics and Prebiotics 3: 271–280. Lahtinen SJ, Davis E, and Ouwehand AC (2012) Lactobacillus species causing obesity in humans: Where is the evidence? Beneficial Microbes 3: 171–174. Nowak A and Libudzisz Z (2009) Ability of probiotic Lactobacillus casei DN 114001 to bind or/and metabolize heterocyclic aromatic amines in vitro. European Journal of Nutrition 48: 419–427. Sanders ME, Akkermans LMA, Haller D, et al. (2010) Safety assessment of probiotics for human use. Gut Microbes 1(3): 164–185. Vankerckhoven V, Huys G, Vancanneyt M, et al. (2008) Biosafety assessment of probiotics used for human consumption: Recommendations from the EUPROSAFE project. Trends in Food Science and Technology 19: 102–114.
Whelan K and Myers CE (2010) Safety of probiotics in patients receiving nutritional support: A systematic review of case reports, randomized controlled trials, and nonrandomized trials. American Journal of Clinical Nutrition 91: 687–703.
Relevant Websites www.ahrq.gov/clinic/tp/probiotictp.htm Agency for Healthcare Research and Quality: Safety of Probiotics to Reduce Risk and Prevent or Treat Disease (2011) Evidence report/Technology assessment No. 200. AHRQ Publication No.11-E007. www.efsa.europa.eu/en/topics/topic/qps.htm EFSA. ftp://ftp.fao.org/es/esn/food/wgreport2.pdf FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food (2002). http://www.SFDA.com State Food and Drug Administration (SFDA) in China.