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Probiotics and Human Health

By: Chloe McClanahan, BioFiles Edition 6.2

At the beginning of the 20th century, probiotics were thought to have a beneficial effect to the host by improving the intestinal microbial balance. Through his studies and observations, the Russian biologist Metchnikoff noted people from cultures which consumed large amounts of fermented milk, generally had higher lifespans. He proposed that lactic-acid bacteria create a more acidic environment as a result of fermentation, thereby suppressing the growth of proteolytic bacteria. Metchnikoff propagated the consumption of sour milk fermented with the Lactobacillus bulgaricus as a means of promoting intestinal balance and overall health.

Today, diverse studies report the benefits of probiotics, such as inhibitory effects on pathogens, aid in the management or prevention of chronic intestinal inflammatory diseases or atopic syndromes, and support to the immune system. Potential beneficial applications abound, researchers continue to evaluate the effictiveness and clarify the mechanisms of action of probiotics. Nevertheless, the use of probiotics has surged due to direct to consumer marketing and lenient regulatory control.

The efficacy of probiotics in shortening the duration of acute infectious diarrhea has been repeatedly confirmed but controlled studies need to be performed to guide the dosage, duration, and strain combination for specific patient groups. Similarly, the use of probiotics in gastrointestinal inflammatory diseases, including ulcerative colitis, Crohn's disease, and irritable bowel syndrome has been investigated but only a few randomized controlled trials have been conducted.

Current research is addressing the modulation of gastrointestinal immune function by probiotics. Evidence from animal model studies shows probiotic treatments provide protection from inflammation of the colon by down regulating inflammatory cytokines or inducing regulatory mechanisms. The examination of probiotic influence on dendritic cell function and cytokine secretion is ongoing. Recently, scientists in Finland have discovered cheese can help preserve and enhance the immune system of the elderly by acting as a carrier for probiotic bacteria. The research showed daily consumption of probiotic cheese helps to bolster immunity in elderly people. Cheese was confirmed to be an effective carrier for probiotics.

The cytoprotective potential of probiotics is also being investigated. A study reported exopolysaccharides released into the surrounding environment by the probiotic strain Bacillus coagulans RK-02 had significant antioxidant and free radical scavenging activity. Another study effectively used a probiotic formulation against acetaminophen induced hepatotoxicity.

Sigma Life Science provides the quality, service, and product selection to support the movement towards a deeper understanding of the relationship between probiotics, intestinal microbiota, and human health.

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Bifidobacterium

Bifidobacterium is one of the most important probiotic bacteria used in the dairy industry. They are Gram-positive, non-motile, rodshaped, and often branched anaerobic bacteria. They were first isolated from a breast-fed infant by Henry Tissier of the Pasteur Institute. At that time, Tissier named the organism Bacillus bifidus communis . Bifidobacteria have a positive effect on the immune system and help to control intestinal pH. In addition, Bifidobacteria produce bacteriocins and bacteriocin-like inhibitory compounds which inhibit the growth of other bacteria.

Bifidobacteria posess many glycosylases able to degrade various plant or milk derived oligosaccharides. Several such enzymes were identified on the Bifidobacterium genome. The genomic sequencing of Bifidobacterium animalis subsp. lactis AD011 revealed a glycosyl hydrolase cluster containing a transcriptional regulator and an ABC transporter, as well as a fos gene cluster that is involved in the processing of prebiotics. Bifidobacteria are able to utilize a broad range of substrates as energy sources, such as plant polymers, glycoproteins, and glycoconjugates, as well as having specialized proteins for the catabolism of oligosaccharides.

Bifidobacteria also have a unique hexose metabolism called the bifid shunt. The key enzyme, fructose-6-phosphate phosphoketolase is not found in any other Gram-positive intestinal bacteria and therefore, provides an ideal target for a diagnostic test.

It was found that live Bifidobacterium lactis can directly counteract the harmful effects of coeliac-toxic gliadin and this may prove to be a future potential treatment of coeliac disease.

In adult intestines, only 3–6% of the fecal flora is composed of bifidobacteria, while in breast-fed infants bifidobacteria can constitute up to 90%. With increasing age, the number of bifidobacteria decreases. It was observed that babies and adults with lower numbers of bifidobacteria have a higher risk for diarrhea and allergies. For this reason, bifidobacteria are added as a probiotic supplement to infant formulas, drinks, yogurts, and a range of other products.

Because of the wide use of bifidobacteria, Sigma-Aldrich has developed a bifidobacteria selective medium (BSM), available as an agar or a broth, as a standard for quality control. This medium allows for fast and easy quality control of yogurt made with bifidobacteria and can be used to control the count of bifidobacteria. Bifidobacterium grow very well on this medium, while Lactobacillus and Streptococcus strains are inhibited. Bifidobacterium colonies grow within 24–48 hours (occasionally up to three days because of the highly selective conditions). The Bifidobacterium colonies are purple-brown, and therefore, are easy to differentiate from other organisms. In a Swiss governmental evaluation study for the enumeration of bifidobacteria in sour milk products, the traditional method was compared to Wilkins-Chalgren Agar with 100 mg/L mupirocine and BSM Agar. The traditional method produced statistically significant differences, while Wilkins-Chalgren Agar and BSM Agar showed similar results without any significant variances.

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Lactobacillus

Lactobacilli are rod-shaped, Gram-positive, fermentative, facultative anaerobic or microaerophilic organotrophs. Normally they form straight rods, but under certain conditions spiral or coccobacillary forms have been observed. In most cases, they form chains of varying length. Lactobacilli belong to the lactic acid bacteria and comprise the major part of this group. As their name implies, they produce lactic acid and derive energy from the fermentation of lactose, glucose, and other sugars to lactate via homofermentative metabolism. About 85–90% of the sugar utilized in the fermentative process is converted to lactic acid. This acid producing mechanism inhibits growth of other organisms and favors the growth of lactobacilli that thrive in low pH environments. ATP is generated during the process by non-oxidative substrate-level phosphorylation.

Some lactobacilli strains were shown to produce, like bifidobacteria, a bacteriocinlike substance and are able to inhibit a broad range of pathogens. Lactobacilli also produce adhesins (proteins), which perform a vital role in recognizing specific host components (extracellular matrix) important for the bacterial adhesion and colonization at host surfaces, as well as in bacterial interaction with physiological and immunological processes.

In the last several years, several new Lactobacillus species have been introduced as probiotics, including Lactobacillus rhamnosus , Lactobacillus casei , and Lactobacillus johnsonii . As of this time, probiotics have not been used in the pharmaceutical industry due to the many open questions that remain to be answered.

Since lactobacilli prefer acidic conditions, natural extracts and juices from tomatoes and oranges, as well as other single metabolic acids (e.g. malic acid), are often used as media ingredients. Casein and yeast extract provide rich amino acid sources, and the maltose is used as a carbohydrate source for lactobacilli, which cannot utilize glucose as fermentable sugar. Fructose is the carbohydrate source of Lactobacillus fructivorans . Polysorbate, sorbitan monooleate and other related compounds act as a source of fatty acids and stimulate the lactic acid bacteria. Today, it is standard practice to differentiate lactobacilli based on their phenotype using selective media. Classical phenotypic tests for identification of lactobacilli are based on physiological characteristics, like motility, growth temperature, respiratory type, and growth in sodium chloride, as well as on diverse biochemical characteristics, such as fermentation type, metabolism of carbohydrate substrates, production of lactic acid isomers, coagulation of milk, and presence of specific enzymes like arginine dihydrolase. In Bergey’s Manual, Lactobacillus is described as a Gram-positive rod, nonspore forming, acid fast negative and catalase negative. The colony morphology on certain media is taken for the presumptive identification.

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Microbiological Media and Detection

Selective Media for Bifidobacteria

Name Application Cat. No.
BSM Agar For the selective isolation and identification of Bifidobacteria.
The medium is used for quality control in the manufacture of dairy products.
88517
BSM Broth For the selective isolation and identification of Bifidobacteria.
The medium is used for quality control in the manufacture of dairy products.
90273
BSM Supplement An antimicrobial supplement recommended for the selective isolation of Bifidobacteria. 83055
Wilkins Chalgren anaerobic agar Used for the isolation of anaerobic bacteria. W1761

 

Differentiation Media for Lactobacilli

Name Application Cat. No.
Litmus Milk For maintenance of Lactobacilli and for determining the action of bacteria on milk. 17158
LS Diff erential Agar For the maximum growth and differentiation of Lactobacilli and Streptococci on the basis of colonial morphology, 2,3,5-triphenyltetrazolium chloride reduction, and casein reaction. 17153
WL Nutrient Agar For the examination of materials encountered in brewing and for industrial fermentations containing mixed fl ora of yeasts and bacteria. 17222
WL nutrient broth Recommended for the cultivation of bacteria encountered in breweries and industrial fermentations. W2261

 

Nonselective Media for Lactobacilli

Name Application Cat. No.
Elliker Broth For culturing Streptococci and Lactobacilli of importance in the dairy industry. 17123
Plate Count Skim Milk Agar For the enumeration of bacteria in milk and dairy products. 80957
Tomato Juice Agar For the cultivation and enumeration of Lactobacilli. 17216
Tomato Juice Broth For cultivation of yeasts and other aciduric microorganisms. 17218
Tryptone Glucose Yeast Extract Agar Recommended for enumeration of bacteria in water, air, milk and dairy products. T2188
Yeast malt agar Used for the isolation and cultivation of yeasts, molds and other aciduric microorganisms. Y3127

 

Selective Media (by low pH and selective agents) for Lactobacilli

Name Application Cat. No.
Lactobacillus bulgaricus Agar (Base) Used with acetate buff er for isolation and identifi cation of Lactobacillus bulgaricus . 69964
MRS Agar For the enrichment, cultivation and isolation of all species of Lactobacillus from all types of material according to DeMan, Rogosa, and Sharpe. 69964
MRS Agar, original acc. DeMan-Rogosa-Sharpe For the enrichment, cultivation and isolation of all species of Lactobacillus from all types of materials. Recommended by the "Schweizerisches Lebensmittelbuch" 5th ed., chapter 56A. 30912
MRS Agar, Vegitone This MRS Agar is free of animal derived material. It is recommended for the isolation and cultivation of Lactobacillus species. 41782
MRS Broth For the enrichment and isolation of all species of Lactobacilli from all types of material. 69966
MRS Broth modified, Vegitone This MRS Broth contains plant peptone instead of animal peptone. It is recommended for the isolation and cultivation of Lactobacillus species. 38944
NBB Agar Base, modified Selective medium used for the detection of contaminating/spoilage microorganisms in brewery. 64198
NBB Broth Base, modified Selective medium used for the detection of contaminating/spoilage microorganisms in brewery. 50725
Orange-serum Agar For the isolation, cultivation and enumeration of acid-tolerant spoilage microorganisms in fruit juice and fruit juice concentrates, in particular from citrus fruit, according to Hays, Troy, and Beisel. The prepared agar may be turbid after autoclaving due to orange extract. 75405
Raka-Ray Agar A medium for selective isolation of lactic acid bacteria from beer and brewing processes 02538
Rogosa SL agar Used as a selective medium for cultivation of Lactobacilli. R1148
Sorbic acid Agar (Base) For the isolation and differentiation of Lactobacilli from food, feces, etc. according to Reuter. 85515
Universal Beer Agar For culturing microorganisms of signifi cance in the brewing industry. 17226
WL Differential Agar For selective isolation and enumeration of bacteria encountered in breweries and industrial fermentations. 17215

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HybriScan™ Detection

Molecular biology-based methods, like PCR, can be used for lactobacilli detection. However, they are often quite expensive. Sigma-Aldrich provides a revolutionary molecular biology method that is rapid, easy, and cost effective. Based on the detection of rRNA, this method completely avoids the need for PCR amplification. The sandwich hybridization test, called HybriScan, is performed on a microtiter plate. The range of lactobacilli detected by HybriScan tests is listed in the table. For more information about the test and the technical principles, please visit sigma-aldrich.com/hybriscan.

 

Name Application Reactions Cat. No.
HybriScan™ D Beer Detection of all relevant beer spoilage organisms of genus Lactobacillus, Pediococcus, Pectinatus and Megasphaera 96 reactions 62533
HybriScan™ D Drinks Detection of bacteria and yeasts in fruit juices and nonalcoholic beverage; determination of total viable count 96 reactions 68301
HybriScan™ D Lactobac Detection of Lactobacilli in fruit juices and non-alcoholic beverage 96 reactions 59744
HybriScan™ I Lactobacillus brevis Identification of Lactobacillus brevis 48 reactions 75724
HybriScan™ I Lactobacillus buchneri Identification of Lactobacillus buchneri 48 reactions 80065
HybriScan™I Lactobacillus lindneri Identification of Lactobacillus lindneri 48 reactions 86827

 

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Fermentation: Enzymes, Substrates, and SCFA

The influence of probiotics on the metabolic activity of intestinal microbiota may be evaulated by detecting bacterial enzymatic activity in fecal samples, specifically β-glucosidase, β-glucuronidase, and urease activity. Experimental studies using probiotics have indicated a significant decrease in β-glucuronidase activity. A decrease in β-glucuronidase decreases the release of carcinogens from carcinogen-glucuronide conjugates within the colonic lumen.

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Enzymes

Name Specific Activity Unit Definition Form Cat. No.
β-Glucosidase from almonds ≥2 units/mg solid One unit will liberate 1.0 μmole of glucose from salicin per min at pH 5.0 at 37 °C. lyophilized powder G0395
β-Glucosidase from almonds 10–30 units/mg solid lyophilized powder G4511
β-Glucuronidase from Escherichia coli ≥20,000 units/mg protein One Sigma or modified Fishman unit will liberate 1.0 μg of phenolphthalein from phenolphthalein glucuronide per hr at 37 °C at pH 6.8 (30 min assay). lyophilized powder G8420
β-Glucuronidase from Escherichia coli 1,000,000–5,000,000 units/g protein lyophilized powder G7396
β-Glucuronidase from Escherichia coli 5,000,000–20,000,000 units/g protein lyophilized powder G7646
β-Glucuronidase from Escherichia coli 20,000,000–60,000,000 units/g protein aqueous glycerol solution G8162
β-Glucuronidase from Escherichia coli 20,000,000–60,000,000 units/g protein lyophilized powder G7896
β-Glucuronidase from Escherichia coli ≥10,000,000 units/g protein lyophilized powder G8295
Urease from Canavalia ensiformis (Jack bean) 15,000–50,000 units/g solid One micromolar unit will liberate 1.0 μmole of NH 3 from urea per min at pH 7.0 at 25 °C. It is equivalent to 1.0 I.U. or 0.054 Sumner unit (1.0 mg ammonia nitrogen in 5 minutes at pH 7.0 at 20 °C) powder U1500
Urease from Canavalia ensiformis (Jack bean) 50,000–100,000 units/g solid powder U4002
Urease from Canavalia ensiformis (Jack bean) ≥600,000 units/g solid powder U0251
Urease from Canavalia ensiformis (Jack bean) - glycerol solution U1875

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Detection Substrates

Glucoside

Name Assay Cat. No.
3-Acetylumbelliferyl β-D -glucopyranoside ≥95% A3582
5-Bromo-4-chloro-3-indolyl β-D-glucopyranoside ≥97% B4527
6-Bromo-2-naphthyl β-D-glucopyranoside ≥99%, TLC B7877
6-Chloro-3-indolyl β-D-glucopyranoside ≥99.0%, HPLC 93546
Fluorescein di-(β-D-glucopyranoside) ≥90% F4521
Indoxyl β-D-glucoside ≥97% I3750
Indoxyl β-D-glucoside ≥97% I6893
2-Methoxy-4-(2-nitrovinyl)phenyl β-D- glucopyranoside ≥95% M2918
4-Methylumbelliferyl α-D-glucopyranoside - M9766
4-Methylumbelliferyl β-D-glucopyranoside - M3633
2-Nitrophenyl β-D-glucopyranoside - N8016
4-Nitrophenyl α-D -glucopyranoside ≥99% N1377
4-Nitrophenyl β-D-glucopyranoside ≥98%, TLC N7006
Resorufin β-D-glucopyranoside ≥90% R4758

 

Glucuronide

Name Assay Cat. No.
5-Bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt ≥98% B3783
5-Bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt ≥98%, TLC B6650
5-Bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt 10 mg substrate per tablet - B8049
5-Bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt ≥98% B0522
5-Bromo-4-chloro-3-indolyl β-D-glucuronide sodium salt ≥98% B5285
5-Bromo-4-chloro-3-indolyl β-D-glucuronide sodium salt 10 mg substrate per tablet - B8174
5-Bromo-6-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt ≥98% B4532
6-Chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt ≥97.0%, HPLC 24907
Indoxyl β-D-glucuronide cyclohexylammonium salt - I7638
4-Methylumbelliferyl β-D-glucuronide hydrate ≥98%, TLC M9130
Naphthol AS-BI β-D-glucuronide - N1875
4-Nitrophenyl β-D-glucuronide ≥98%, TLC N1627
Phenolphthalein β-D-glucuronide - P0501
Phenolphthalein β-D-glucuronide sodium salt - P0376
4-Trifl uoromethylumbelliferyl glucuronide potassium salt ≥98%, TLC T6410

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Short-Chain Fatty Acids (SCFA)

Probiotics and prebiotics enhance SCFA end product synthesis. An increase in SCFA production lowers the gastrointestinal pH thus improving pathogen resistance and stimulating epithelial cell proliferation. The predominant SCFA, butyrate, is the preferred substrate for colonocytes which likely contributes to a normal colonocyte population and a lower cancer risk. SCFA production can be measured with gas chromatography analysis on fecal samples or intestinal modeling systems. However, due to normal colon absorption of 95% of the SCFA metabolites, stable isotope technology is a superior method of analysis.

Non-Labeled Short-Chain Fatty Acids

Name Cat. No.
Acetic acid, ReagentPlus®, ≥99% A6283
Butyric acid, ≥99% B103500
Hexanoic acid, 99% H12137
Isobutyric acid, 99% I1754
Isovaleric acid, 99% 129542
Propionic acid, reagent grade, ≥99% P1386
Valeric acid, ≥99% 240370

Isotec® Short-Chain Fatty Acid Isotopes

Name Cat. No.
Sodium acetate-1-13C, S & P tested, 99 atom % 13C 668656
Sodium acetate-1-13C, 99 atom % 13C 279293
Sodium butyrate-1-13C, 99 atom % 13C 292656
Sodium propionate-1-13C, 99 atom % 13C 279455

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Bile Salts and Bile Salt Hydrolase

Several Lactobacillus and Bifidobacterium probiotic strains exhibit bile salt hydrolase (BSH) activity and are suspected to play a role in combating the negative effects of bile via detoxification of bile salts. However, enhanced BSH activity is potentially detrimental to the host. More research is needed to understand this dynamic between probiotics and bile salts.

Name Cat. No.
Bile bovine, dried, unfractionated B3883
Bile from bovine and ovine B8381
Bile salts mixture B3426
Sodium chenodeoxycholate, ≥97% C8261
Sodium cholate hydrate, from ox or sheep bile, ≥99% C1254
Sodium deoxycholate, ≥97% (titration) D6750
Sodium deoxycholate monohydrate, BioXtra, ≥99.0% (titration) D5670
Sodium glycochenodeoxycholate, ≥97% (TLC) G0759
Sodium glycocholate hydrate, ≥97% (TLC) G7132
Sodium glycodeoxycholate, BioXtra, ≥97% (TLC) G9910
Sodium taurochenodeoxycholate T6260
Sodium taurodeoxycholate hydrate, BioXtra, ≥97% (TLC) T0557
Taurocholic acid sodium salt hydrate, ≥95% (TLC) T4009

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Materials

     

 References

  1. Vaughan, R.B., The romantic rationalist: A study of Elie Metchnikof. Medical History , 9 , 201-215 (1965).
  2. Verna, E.C., Use of probiotics in gastrointestinal disorders: what to recommend? Therap. Adv. Gastroenterol. , 3 , 307-319 (2010).
  3. Borchers, A.T. et al., Probiotics and immunity. J. Gastroenterol. , 44 , 26-46 (2009).
  4. Ibrahim, F. et al., Probiotics and immunosenescence: cheese as a carrier. FEMS Immunology & Medical Microbiology , 59 , 53-59 (2010).
  5. Kodali, V.P. and Sen, R. Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium. Biotechnol. J. , 3 , 245-251 (2008).
  6. Sharma, S. et al. Modulation of Bax/Bcl-2 and caspases by probiotics during acetaminophen induced apoptosis in primary hepatocytes. Food Chem. Toxicol. (2010). [Epub ahead of print].
  7. De Preter, V. et al., The impact of pre- and/or probiotics on human colonic metabolism: Does it affect human health? Mol. Nutr. Food Res. , 54 , 1-12 (2010).
  8. Topping, D.L. and Clifton, P.M., Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Phys. Rev. , 81 , 1031- 1064 (2001).
  9. Chow, J. Probiotics and prebiotics: A brief overview. J. Ren. Nutr. , 12 , 76-86 (2002).
  10. Backhed, F. et al., Host-bacterial mutualism in the human intestine. Science , 307 , 1915-1920 (2005).
  11. Mikkelsen, L.L. et al., Enumeration of bifidobacteria in gastrointestinal samples from piglets. Appl. Environ. Microbiol. 69 , 654-658 (2003).
  12. Rada, V. and Petr, J., A new selective medium for the isolation of glucose non-fermenting bifi dobacteria from hen caeca. J. Microbiol. Methods, 43 127-132 (2000).
  13. Grand, M. et al., Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products. Eur. Food Res. Technol. 217 , 90-92 (2003).
  14. Rada, V. and Koc, J., The use of mupirocin for selective enumeration of bifidobacteria in fermented milk products. Milchwissenschaft., 55 , 65-67 (2000).
  15. Federal Offi ce of Public: Swiss Food Manual, Chapter 56, Microbiology, Neuausgabe 2000, Stand (2004).
  16. IUPAC: Protocol for the design, conduct and interpretation of method-performance studies.
  17. Nowroozi, J. et al., Study of Lactobacillus as Probiotic Bacteria, Iranian J. Publ. Health , 33(2) , 1-7 (2004).
  18. Tissier, H., Recherchers sur la fl ora intestinale normale et pathologique du nourisson. Thesis, University of Paris (1900).
  19. Tannock, G.W., Probiotics: time for a dose of realism. Curr. Issues Intest. Microbiol. , 4(2) , 33-342 (2003).
  20. Ljungh, Å. and Wadström, T., Lactobacillus . Molecular Biology: From Genomics to Probiotics, Caister Academic Press (2009).
  21. Lindfors, K et al., Live probiotic Bifidobacterium lactis bacteria inhibit the toxic effects induced by wheat gliadin in epithelial cell culture. Clin. Exp. Immunol. , 152(3) , 552-558 (2008).
  22. Kim, J.F. et al., Genome sequence of the probiotic bacterium Bifidobacterium animalis subsp. lactis AD011, J. Bacteriol. , 191(2) , 678-679 (2009).
  23. Kajander, et al., Eff ects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome., Aliment. Pharmacol. Ther. , 26 , 463- 473 (2007).
  24. Burns, A.J. and Rowland, I.R. Anti-carcinogenicity of probiotics and prebiotics. Curr. Issues Intest. Microbiol. , 1(1) , 13-24 (2000).
  25. Begley, M. et al., Bile salt hydrolase activity in probiotics. Appl. Environ. Microbiol. , 72(3) , 1729-1738 (2006).
  26. Janda, M.J. and Abbott, S.L, 16S rRNA Gene Sequencing for Bacterial Identifi cation in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. J. Clin. Microbiol. , 45(9) , 2761-2764 (2007).
  27. Matsuki, T. et al., Quantitative PCR with 16S rRNAGene- Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria . Appl. Environ. Microbiol. , 70(1) , 167-173 (2004).

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