International Dairy Journal 17

Review

Keywords: L. acidophilus; Bifidobacterium; L. casei; Health benets; Stability; Bioactive peptides

3.6. Reduction in serum cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.7. Helicobacter pylori infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

1. Introduction

ARTICLE IN PRESSAt the beginning of this century, Nobel Laureate ElieMetchnikoff, at the Pasteur Institute, linked health and

0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.idairyj.2007.01.014

Tel.: +61 3 9919 8289; fax: +61 3 9919 8284.E-mail address: Nagendra.Shah@vu.edu.au.3.8. Inammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.9. Immune system stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267

4. Prebiotics and synbiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268

5. Alternate products for incorporating probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268

5.1. Products based on bioactive peptides released from milk proteins by proteolytic probiotics . . . . . . . . . . . . . . . . . . 1271

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1273

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1273Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262

2. Probiotic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263

3. Health benets of functional probiotic cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264

3.1. Antimicrobial activity and gastrointestinal infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264

3.2. Effectiveness against diarrhoea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3.3. Improvement in lactose metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3.4. Antimutagenic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3.5. Anticarcinogenic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266probiotics.

r 2007 Elsevier Ltd. All rights reserved.Functional cultures and health benets

Nagendra P. Shah

School of Molecular Sciences, Victoria University, P.O. Box 14428, Melbourne, Vic. 8001, Australia

Received 25 September 2006; accepted 22 January 2007

Abstract

A number of health benets have been claimed for probiotic bacteria such as Lactobacillus acidophilus, Bifidobacterium spp., and

L. casei. These benets include antimutagenic effects, anticarcinogenic properties, improvement in lactose metabolism, reduction in

serum cholesterol, and immune system stimulation. Because of the potential health benets, these organisms are increasingly being

incorporated into dairy foods, particularly yoghurt. In addition to yoghurt, fermented functional foods with health benets based on

bioactive peptides released by probiotic organisms, including Evoluss and Calpiss, have been introduced in the market. To maximize

effectiveness of bidus products, prebiotics are used in probiotic foods. Synbiotics are products that contain both prebiotics and(2007) 12621277

www.elsevier.com/locate/idairyj

1900s that scientists began to investigate the reasons forthose benets. A number of genera of bacteria (and yeast)are used as probiotics, including Lactobacillus, Leuconos-toc, Pediococcus, Bifidobacterium, and Enterococcus, butthe main species believed to have probiotic characteristicsare L. acidophilus, Bifidobacterium spp., and L. casei.Members of the genera Lactobacillus and Bifidobacteriumhave a long and safe history in the manufacture of dairyproducts and are also found as a part of gastrointestinalmicroora. Probiotic bacteria with desirable properties andwell-documented clinical effects include L. johnsonii La1,L. rhamnosus GG (ATCC 53103), L. casei Shirota,L. acidophilus NCFB 1478, B. animalis Bb12 and L. reuteri(Shah, 2004).At present, 56 species of the genus Lactobacillus have

been recognized (Table 1). L. acidophilus is the mostcommonly suggested organism for dietary use. Growth ofL. acidophilus occurs at as high as 45 1C, however, theoptimum growth temperature is between 3540 1C. Theorganisms grow in slightly acidic media at pH of 6.44.5,but growth ceases when a pH of 4.03.6 is reached. Theacid tolerance of the organisms varies from 0.3% to 1.9%

ARTICLE IN PRESSiry Journal 17 (2007) 12621277 1263longevity to ingestion of bacteria present in yoghurt(monograph reprinted in: Metchnikoff, 2004). It wasbelieved that bacteria present in yoghurt controlledinfections caused by enteric pathogens and regulatetoxaemia, both of which play a major role in ageing andmortality. This observation provided a major boost to themanufacture and consumption of yoghurt. The healthbenets derived by the consumption of foods containingLactobacillus acidophilus, Bifidobacterium and L. casei arenow well documented.Functional foods are dened as foods that contain some

health-promoting component(s) beyond traditional nutri-ents (Shah, 2001). Functional foods are also known asdesigner foods, medicinal foods, nutraceuticals, therapeuticfoods, superfoods, foodiceuticals, and medifoods (Shah,2001). In general, the term refers to a food that has beenmodied in some way to become functional. One way inwhich foods can be modied to become functional is byaddition of probiotics: the word probiotic originated fromGreek meaning for life. Probiotic foods are dened asfood containing live microorganisms believed to activelyenhance health by improving the balance of microora inthe gut (Fuller, 1992). Probiotic yoghurts, for instance,contain probiotic bacteria as health promoting componentsbeyond traditional nutrients.Traditionally, yoghurt is manufactured using Strepto-

coccus thermophilus and L. delbrueckii ssp. bulgaricus asstarter cultures. These organisms are claimed to offer somehealth benets as postulated by Metchnikoff; however,they are not natural inhabitants of the intestine. Therefore,for yoghurt to be considered as a probiotic product,L. acidophilus, Bifidobacterium and L. casei are incorpo-rated as dietary adjuncts. Products such as Yakult containthe L. acidophilus Shirota strain. Whilst fermented milkproducts containing only one or more of these threeadjuncts could be manufactured, the longer incubationperiod required and poorer resultant product quality arethe two main factors that preclude such practice commer-cially. Thus, the normal practice is to make productwith both starter organisms, e.g., Str. thermophilus andL. delbrueckii ssp. bulgaricus, and one or more species ofprobiotic bacteria.New fermented products containing L. acidophilus,

Bifidobacterium spp., L. casei Shirota, L. rhamnosus GG,and L. reuteri have been developed in Europe. However,L. acidophilus and Bifidobacterium spp. are most commonlyused as probiotics. It is estimated that over 70 productscontaining L. acidophilus and Bifidobacterium spp., includ-ing yogurt, buttermilk, frozen desserts and milk powder,are produced worldwide. Probiotic organisms themselvesare also available as powders, capsules and tablets.

2. Probiotic bacteria

Probiotics have been consumed in foods such as yoghurt

N.P. Shah / International Dafor perhaps thousands of years, and while the cultureswere thought to have benecial effects, it was not until thetitratable acidity, with an optimum pH at 5.56.0 (Curry &Crow, 2003; Shah, 2003).L. acidophilus tends to grow slowly in milk, leading to

the risk of overgrowth of undesirable microorganisms.Ironically, most strains of L. acidophilus do not survive wellin fermented milk due to the low pH, and it is difcult tomaintain large numbers in the product. The poor growth ispartly related to low concentration of small peptides andfree amino acids in milk, which would be insufcient tosupport the bacterial growth.

Table 1

Lactobacilli used as probiotic culturesa

Species Strains

L. acidophilus LA-1/LA-5 (Chr. Hansen)

L. acidophilus NCFM (Rhodia)

L. acidophilus Johsonii La1 (Nestle)

L. acidophilus DDS-1 (Nebraska Cultures)

L. acidophilus SBT-2062 (Snow Brand Milk Products)

L. bulgaricus Lb12

L. lactis L1A (Essum AB)

L. casei Immunitas (Danone)

L. plantarum 299v, Lp01

L. rhamnosus GG (Valio)

L. rhamnosus GR-1 (Urex Biotech)

L. rhamnosus LB21 (Essum AB)

L. reuteri SD2112/MM2 (Biogaia)

L. rhamnosus 271 (Probi AB)

L. plantarum (Probi AB)

L. reuteri SD2112 (also known as MM2)

L. casei Shirota (Yakult)

L. paracasei CRL 431 (Chr. Hnasen)

L. fermentum RC-14 (Urex Biotech)

L. helveticus B02aAdapted from Krishnakumar and Gordon (2001), Holm (2003), Playne

et al. (2003); Shah (2004).

Bifidobacterium are normal inhabitants of the humangastrointestinal tract. Recent in vivo scientic studies usinganimals or human volunteers have shown that consump-tion of live Bifidobacterium has an effect on the gutmicroora. Selected strains survive stomach and intestinaltransit and reach the colon in abundant numbers. New-borns are colonized with Bifidobacterium within daysafter birth and the population appears to be relativelystable until advanced age, when a decline in their numbersoccurs. However, diet, antibiotics, and stress are reportedto inuence the population of Bifidobacterium in theintestines.Presently, there are 29 species in the genus Bifidobacter-

ium (Table 2), 14 of which are isolated from human sources(i.e., dental caries, faeces and vagina), 12 from animalintestinal tracts or rumen, and 3 from honeybees.Bifidobacterium species found in humans are: B. adolescen-tis, B. angulatum, B. bifidum, B. breve, B. catenulatum,B. dentium, B. infantis, B. longum, and B. pseudocatenula-tum. B. breve, B. infantis, and B. longum are found inhuman infants; B. adolescentis, and B. longum are found in

ARTICLE IN PRESSN.P. Shah / International Dairy J1264human adults (Shah & Lankaputhra, 2002).The optimum pH for the growth of Bifidobacterium is

6.07.0, with virtually no growth at pH 4.55.0 and belowor at pH 8.08.5 and above. Optimum growth occurs at atemperature of 3741 1C, the minimum and maximumgrowth temperatures are 2528 and 4345 1C, respectively.The main probiotic organisms that are currently used

worldwide belong to the genera Lactobacillus and Bifido-bacterium. A limited number of investigations have alsobeen carried out into the potential properties of generaincluding Pediococcus, Leuconostocs, and Propionibacter-ium and also of Enterococcus faecium. Ent. faecium is

Table 2

Bifidobacterium cultures used as probiotic culturesa

Species Strains

B. adolescentis ATCC 15703, 94-BIM

B. longum BB536 (Morinaga Milk Industry)

B. longum SBT-2928 (Snow Brand Milk

Products)

B. breve Yakult

B. bifidus Bb-11

B. lactis (reclassied as B.

animalis)

Bb-12 (Chr. Hansen)

B. essensis Danone (Bioactivia)

B. lactis Bb-02

B. infantis Shirota

B. infantis Immunitass

B. infantis 744

B. infantis 01

B. laterosporus CRL 431

B. lactis LaftiTM, B94 (DSM)

B. longum UCC 35624 (UCCork)

B. lactis DR10/HOWARU DaniscoaAdapted from Krishnakumar and Gordon (2001); Holm (2003); Playne

et al. (2003); Shah (2004).more pH stable than L. acidophilus and producesbacteriocins against some enteropathogens. These proper-ties make this organism attractive as a probiotic. Frompublished reviews, four strains with the most publishedclinical data are L. rhamnosus GG, L. casei Shirota,B. animalis Bb-12, and Saccharomyces cerevisiae Boulardii(Shah, 2006b).

3. Health benets of functional probiotic cultures

A number of health benets are claimed in favourof products containing probiotic organisms includingantimicrobial activity and gastrointestinal infections, im-provement in lactose metabolism, antimutagenic proper-ties, anticarcinogenic properties, reduction in serumcholesterol, anti-diarrhoeal properties, immune systemstimulation, improvement in inammatory bowel diseaseand suppression of Helicobacter pylori infection (Kurmann& Rasic, 1991; Shah, 2000b, 2004). Some of the healthbenets are well established, while other benets haveshown promising results in animal models. However,additional studies are required in humans to substan-tiate these claims. Health benets imparted by probioticbacteria are strain specic, and not species- or genus-specic. It is important to note that no strain will pro-vide all proposed benets, not even strains of the samespecies, and not all strains of the same species will beeffective against dened health conditions. The strainsL. rhamnosus GG (Valio), S. cerevisiae Boulardii (Bioco-dex), L. casei Shirota (Yakult), and B. animalis Bb12(Chr. Hansen) have the strongest human health efcacydata with respect to management of lactose malabsorp-tion, rotaviral diarrhoea, antibiotic-associated diarrhoea,and Clostridium difficile diarrhoea (Playne, Bennet, &Smithers, 2003; Shah, 2006a, b). There is sufcient evi-dence to support the view that oral administration ofLactobacillus and Bifidobacterium is able to restore thenormal balance of microbial populations in the intestine(Shah, 2006b).

3.1. Antimicrobial activity and gastrointestinal infections

Probiotic bacteria produce organic acids, hydrogenperoxide and bacteriocins as antimicrobial substances thatsuppress the multiplication of pathogenic and putrefyingbacteria. Lactic and acetic acids account for over 90% ofthe organic acids produced. Lowering of pH due to lacticacid or acetic acid produced by these bacteria in the gut hasa bacteriocidal or bacteriostatic effect. Both Bifidobacter-ium and L. acidophilus show antagonistic effects towardsenteropathogenic Escherichia coli, Salmonella typhimurium,Staphylococcus aureus and Cl. perfringens. L. acidophilusproduces various bacteriocins and antibacterial substancessuch as Lactocidin, Acidolin, Acidophilin, Lactacium-Band inhibitory protein. Similarly, Bifidobacterium produces

ournal 17 (2007) 12621277Bidolin and Bilong, which inhibit several pathogenicbacteria (Shah, 1999).

ARTICLE IN PRESSiry J3.2. Effectiveness against diarrhoea

A major problem associated with antibiotic treatment isappearance of diarrhoea, often caused by Cl. difficile. Thisorganism is found in small numbers in the healthyintestine, but disruption of indigenous microora due toantibiotic treatment leads to an increase in their numberand toxin production, which causes the developmentof diarrhoea. Treatment with metonidazole or vancomycinis usually effective but recurrences are common. Probioticshave proved to be useful as a prophylactic regimenwith antibiotic-associated diarrhoea as well as for treat-ment after onset of antibiotic induced diarrhoea. A dailydose of Lactobacillus GG has been found to be effectivein termination of diarrhoea. Studies with a yeast prepara-tion containing S. cerevisiae Boulardii has also beeneffective in treatment of Cl. difficile related colitis (Shah,2004, 2006b).Rotavirus is one of the most common causes of acute

diarrhoea in children worldwide. During diarrhoeal stageof infection, the permeability of gut epithelial cells isincreased to intact proteins. Probiotics are claimed toshorten duration of rotavirus diarrhoea in children(Saavedra, Bauman, Oung, Perman, & Yolken, 1994).The strongest evidence of a benecial effect of denedstrains of probiotics has been established usingL. rhamnosus GG and B. lactis Bb-12 (now reclassied asB. animalis Bb-12) for prevention and treatment ofdiarrhoea and acute diarrhoea in children mainly causedby rotaviruses. Selected probiotic strains are also effectiveagainst antibiotic-associated diarrhoea. Certain probioticstrains can inhibit the growth and adhesion of a range ofenteropathogens. Studies have indicated benecial effectsagainst pathogens such as Sal. typhimurium and Sal.enteriditis. B. longum SBT-2828 has shown inhibition ofenterotoxigenic Escherichia coli. A pediatric beveragecontaining a mix of B. animalis, L. acidophilus, andL. reuteri has been found to be useful in the preventionof rotavirus diarrhoea (Guandalini et al., 2000).L. rhamnosus GG has been reported to be more effective

in treatment of rotavirus diarrhoea than preparationscontaining Str. thermophilus and L. delbrueckii ssp.bulgaricus. L. reuteri has also been effective in shorteningduration of rotavirus diarrhoea. It reduces the duration ofdiarrhoea in children suffering from rotavirus diarrhoea.Treatment with Lactobacillus GG was associated withenhancement of IgA-specic antibody-secreting cells torotavirus and serum IgA antibody level.There is also strong evidence that probiotic strains can

prevent travellers diarrhoea (Hilton, Kolakowski, Singer,& Smith, 1997), which is caused by bacteria, particularlyenterotoxigenic E. coli. Several studies have been carriedout to assess the effects of probiotic preparations asprophylaxis for travellers diarrhoea; however, the resultshave been conicting. In one study, Danish tourists on

N.P. Shah / International Daa 2-week trip to Egypt were given a mixture of livefreeze-dried preparation of L. acidophilus, B. animalis,L. delbrueckii ssp. bulgaricus and Str. thermophilus at adaily dose of 109 cfu. The administration of probioticpreparation reduced the frequency of diarrhoea. A similarstudy conducted with Finnish tourists using a lyophilizedpreparation of Lactobacillus GG also showed a reductionin the occurrence of travellers diarrhoea. On the otherhand, Katelaris, Salam, and Farthing (1995) and de diosPozo-Olano, Warram, Gomez, and Cacazos (1978) havefound no effect in people suffering from travellersdiarrhoea when L. fermentum KLD L. acidophilus andL. bulgaricus were given in separate studies.Yoghurt containing B. longum was found to be effective

in reducing the course of erythromycin induced diarrhoea.Faecal counts of Lactobacillus GG indicated that theorganisms colonized the intestine despite erythromycintreatment. Probiotic reparations containing 4 109 cfu ofB. animalis Bb-12 and L. acidophilus La-5 has shownsimilar results when volunteers received ampicillin alongwith probiotic preparation. Several studies have shown areduction in diarrhoea in subjects taking S. cerevisiaeBoulardii during the period of antibiotic treatment (Shah,2004, 2006a).

3.3. Improvement in lactose metabolism

Relief of the symptoms of lactose malabsorption isprobably the most widely accepted health benet ofprobiotic organisms. Lactose malabsorption is a conditionin which lactose, the principal carbohydrate of milk, is notcompletely hydrolysed into its component monosacchar-ides, glucose and galactose. Since lactose is cleaved into itsconstituent monosaccharides by the enzyme b-D-galactosi-dase, lactose malabsorption results from a deciency of thisenzyme. Lactose malabsorbers often complain of gastricdistress after consuming fresh, unfermented milk or milkproducts due to the formation of hydrogen gas bymicrobial action on undigested lactose in the gut (Shah,1993; Shah, Fedorak, & Jelen, 1992).The traditional cultures used in making yoghurt (i.e.,

L. delbrueckii ssp. bulgaricus and Str. thermophilus) containsubstantial quantities of b-D-galactosidase (Shah, 2000c),and so both yoghurt and probiotic yoghurt are toleratedwell by lactose malabsorbers. However, reduced levels oflactose in fermented products due to partial hydrolysis oflactose during fermentation are only partly responsible forthis greater tolerance for yoghurt. The slower gastricemptying of both semi-solid and pasteurized yoghurtresults in better absorption of lactose (in the latter, enzymeactivity and bacteria are destroyed due to heat treatment;Shah et al., 1992).

3.4. Antimutagenic properties

An antimutagenic effect of fermented milks has beendetected against a range of mutagens and promutagens in

ournal 17 (2007) 12621277 1265various test systems based on microbial and mammaliancells. Probiotic organisms are reported to bind mutagens

ARTICLE IN PRESSry Jto the cell surface (Orrhage, Sillerstrom, Gustafsson,Nord, & Rafter, 1994). Probiotic bacteria are reportedto reduce faecal enzymatic activities including b-glucur-onidase, azoreductase, and nitroreductase, which areinvolved in activation of mutagens (Goldin & Gorbach,1984b).Lankaputhra and Shah (1998) studied the antimutagenic

activity of organic acids produced by probiotic bacteriaagainst several mutagens and promutagens. The TA-100mutant of Sal. typhimurium (His) strain was used as amutagenicity indicator organism. The mutagenicity testwas carried out using the Ames Salmonella test. In theirstudy, butyric acid showed a broad-spectrum antimuta-genic activity against all mutagens or promutagens studiedand live bacterial cells showed higher antimutagenicitythan killed cells. Inhibition of mutagens and promutagensby probiotic bacteria was permanent for live cells andtemporary for killed cells. The results emphasized theimportance of consuming live probiotic bacteria and ofmaintaining their viability in the intestine to provideefcient inhibition of mutagens. Table 3 summarizesstudies pertaining to antimutagenic properties of functionalmicroorganisms.

3.5. Anticarcinogenic properties

Bacteria and metabolic products such as genotoxiccompounds (nitrosamine, heterocyclic amines, phenoliccompounds, and ammonia) are responsible for colorectalcancer. The consumption of cooked red meat, especiallybarbequed meat, and low consumption of bre arereported to play a major role in causing colorectal cancer.The colonic ora are also reported to cause carcinogenesismediated by microbial enzymes such as b-glucuronidase,azoreductase, and nitroreductase, which convert procarci-nogens into carcinogens.Certain strains of L. acidophilus and Bifidobacterium spp.

are reported to decrease the levels of enzymes such asb-glucuronidase, azoreductase, and nitroreductase respon-sible for activation of procarcinogens and consequen-tly decrease the risk of tumour development (Yoon,Benamouzig, Little, Francois-Collange, & Tome, 2000).Short chain fatty acids produced by L. acidophilus andBifidobacterium, L. plantarum and L. rhamnosus arereported to inhibit the generation of carcinogenic productsby reducing enzyme activities (Cenci, Rossi, Throtta, &Caldini, 2002).The anticarcinogenic effect of probiotic bacteria is

reported to be due to the result of removal of sources ofprocarcinogens (or the enzymes that lead to their forma-tion) improvement in the balance of intestinal microora,normalized intestinal permeability (leading to preventionor delaying of toxin absorption), strengthening of intestinalbarrier mechanisms, and activation of non-specic cellularfactors (such as macrophages and natural killer cells) via

N.P. Shah / International Dai1266regulation of g-interferon production. Orally administeredBifidobacterium is also reported to play a role in increasingproduction of IgA antibodies and functions of Peyerspatch cells (Singh et al., 1997). Table 4 summarizes studiespertaining to anticarcinogenic properties of functionalmicroorganisms.

3.6. Reduction in serum cholesterol

The level of serum cholesterol is a major factor forcoronary heart disease, and elevated levels of serumcholesterol, particularly LDL-cholesterol, have been linkedto an increased risk (Liong & Shah, 2006). There is a highcorrelation between dietary saturated fat or cholesterolintake and serum cholesterol level. Feeding of fermentedmilk containing very large numbers of probiotic bacteria(109 bacteria g1) to hypercholesterolaemic human sub-jects has resulted in lowering cholesterol from 3.0 to1.5 gL1.Probiotic bacteria are reported to de-conjugate bile salts:

deconjugated bile acid does not absorb lipid as readily asits conjugated counterpart, leading to a reduction incholesterol level. L. acidophilus is also reported to takeup cholesterol during growth and this makes it unavailablefor absorption into the blood stream (Klaver & Meer,1993). Studies showing reduction in serum cholesterol aresummarized in Table 5.

3.7. Helicobacter pylori infection

Helicobacter pylori is a pathogenic bacterium that causespeptic ulcers, type B gastritis and chronic gastritis and isnormally present in the stomach as an opportunisticpathogen without causing any symptoms (Armuzzi et al.,2001; Sakamoto et al., 2001).Antibiotic treatments can successfully eradicate

H. pylori. However, antibiotics often cause side effectsand make the bacteria more antibiotic resistant. Probioticorganisms do not appear to eradicate H. pylori, but theyare able to reduce the bacterial load in patients infectedwith H. pylori. L. johnsonii La1 and L. gasseri OLL2716have been found to reduce H. pylori colonization andinammation (Felley et al., 2001). Similarly, L. caseiShirota and L. acidophilus are reported to inhibit thegrowth of H. pylori (Cats et al., 2003). Studies showingprobiotic micro-organisms and H. pylori infection aresummarized in Table 6.

3.8. Inflammatory bowel disease

Inammatory bowel disease (ulcerative colitis andCrohns disease) is related to the intestinal microora.Symptoms of inammatory bowel disease include adisturbance in bowel habits and mucosal inammation.In the intestine of people with inammatory bowel diseasethe numbers of Lactobacillus and Bifidobacterium are lowerand those of coccoids and anaerobes are higher. Probiotics

ournal 17 (2007) 12621277do not cure the disease, but once patients are in remissionthrough treatment with corticosteroids, some probiotics

ARTICLE IN PRESSiry JTable 3

Antimutagenic properties of functional microorganisms

Microorganism Antimutagenic activity

N.P. Shah / International Dacan prolong the remission period, thus reducing theincidence of relapse and the use of corticosteroids. Thisimproves the quality of life of patients. Studies showingprobiotic micro-organisms and inammatory bowel diseaseare summarized in Table 7.

L. acidophilus strains Microbial binding leading

such as 4-nitroquinoline-N

L. acidophilus 2400, 2401, 2404, 2405, 2409,

2415; B. bifidum 1900, 1901, 1912, 1920, 1930

and 1941

Antimutagenic activity of

against mutagens and prom

and 2-amino-3-methyl-3H

L. casei CRL 431 The immune cells activated

mice were those of the inn

specic markers of these c

modication in the numb

L. rhamnosus GG Inuenced intestinal phys

modulation of the endoge

L. acidophilus (LAFTIs L10); B. lactis (LAFTI

B94)

Synbiotic combinations in

induced damage to coloni

L. acidophilus (SBT0274, SBT1703, SBT10239,

SBT10241); B. longum (SBT 2928)

Exhibited a high percentag

amino acid pyrolysates us

of Sal. typhimurium TA 9

L. bulgaricus 291; Str. thermophilus F4, V3; B.

longum BB536

Clear evidence for DNA-p

yoghurt production again

which are targets of tumo

nitrosoguanidine, 1,2-dim

azoxymethane and also re

pre-carcinogenic lesions in

Lactobacillus GG; L. delbrueckii subsp.

rhamnosus (LGG); B. lactis Bb12

The groups treated with p

the total number of tumou

(P 0.04) than in untreatprotective effect of probio

L. helveticus L89 Antimutagenic compound

by L. helveticus, and the r

contributing mechanism. H

variant of the same strain

nitroquinoline-N0-oxide (4

B. infantis CCRC 14633, B. longum B6,

L. acidophilus CCRC 14079 and Str.

thermophilus CCRC 14085

Unfermented soymilk exer

dimethyl-4-amino-bipheny

NQO), the fermented soy

against DMAB than 4-NQ

themophilus and B. infanti

antimutagenicity of 85.07%

and DMAB.

L. acidophilus LA 106 The milk cultured with L.

inhibition of 77% against

nitrosoguanidine among t

Probiotic bacterium Ent. faecium M-74 Exerted different antimuta

N0-nitro-N-nitrosoguanidiinduced mutagenicity in S

presence (+Se) or absence

antimutagenicity of MRS(

Se) extract. Selenium also

and killed cells of Ent. faeReferences

ournal 17 (2007) 12621277 12673.9. Immune system stimulation

The intestine is the bodys largest immune organ and theintestinal microora and the metabolic activity of theintestine is equivalent to that of the liver. Probiotics may

to inhibition of mutagens and promutagens0-oxide, 2-nitrouorene and benzopyrene.

Ayebo et al. (1982)

organic acids produced by probiotic bacteria

utagens such as 2-nitroourene, aatoxin-B,

-imidazoquinoline.

Lankaputhra and Shah

(1998)

after oral L. casei administration to BALB/c

ate immune response (with an increase in the

ells, CD-206 and TLR-2), with no

er of T cells.

Galdeano and Perdigon

(2006)

iology either directly or indirectly through

nous ecosystem or immune system.

Marteau et al. (2001)

regulating the consequences of carcinogen-

c epithelial cells in male Sprague-Dawley rats.

Le Leu et al. (2005)

e of antimutagenicity and binding in vitro on

ing a streptomycin-dependent (SD510) strain

8.

Sreekumar and Hosono

(1998)

rotective effects of lactobacilli used for

st DNA damage caused by HCAs in organs

r induction by 1-methyl-3-nitro-1-

ethylhydrazine, N-methyl-N-nitrosourea and

duce chemically induced-DNA migration and

colon cells of male Fischer 344 rats.

Zsivkovits et al. (2003);

Wollowski et al. (2001)

robiotics, the proportion of cancers relative to

rs in the same groups was signicantly lower

ed male F344 rats suggesting that the possible

tics is restricted to malignant tumours.

Femia et al. (2002)

s were produced in milk during fermentation

elease of peptides was one possible

owever, milk fermented by a non-proteolytic

showed no inhibitory effects on 4-

-NQO).

Matar et al. (1997)

ted lower antimutagenic activity against 3, 20-l (DMAB) than 4-nitroquinoline-N-oxide (4-

milk, showed a higher antimutagenic activity

O. Soymilk fermented with both Str.

s simultaneously exhibited the highest

and 85.78%, respectively, against 4-NQO

Hsieh and Chou (2006)

acidophilus LA 106 (LA2) showed the highest

the mutagenicity of N-methyl-N-nitro-N-

he 71 strains tested.

Hosoda et al. (1992)

genic activity against ooxacin-, N-methyl,

ne- and sodium 5-nitro-2-furylacrylate-

al. typhimurium assay depending on the

of disodium selenite pentahydrate (-Se). The

+Se) extract was higher than that of MRS(-

enhanced the antimutagenic effect of both live

cium M-74, respectively.

Belicova et al. (2004)

ARTICLE IN PRESS

ion

t-b

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the

FM

ts h

, da

ld r

uc

bio

oxi

tab

n c

e a

ucta

ry JTable 4

Probiotic microorganisms and their anticarcinogenic properties

Microorganisms Anticarcinogenic funct

L. acidophilus NCFM Rats consuming a mea

cancer after a 20-wk e

supplement increased

Administration of NC

tumours. NCFM fed ra

the faeces.

L. acidophilus NCFM Using human subjects

resulted in a 2- to 4-fo

glucuronidase, nitrored

L. bulgaricus; Str. thermophilus Ingestion of viable pro

detoxication of genot

L. bulgaricus; Str. thermophilus Possibly inuenced me

the colon.

Lactobacillus and Bifidobacterium; cellular

components and metabolites of LAB

Limits DNA damage i

Bifidobacterium fermented milk; fermented milk

with L. acidophilus, B. bifidum, Lc. lactis ssp.

lactis, Lc. lactis ssp. cremoris

Procarcinogenic enzym

nitroreductase, azored

N.P. Shah / International Dai1268directly or, by changing the composition or activity of theintestinal microora, indirectly inuence the bodys im-mune function (Marteau et al., 1997). Probiotic culturesproduce g-interferon by T-cells and stimulate cytokines asrepresented by TNF-a (tumour necrosis factor) and IL-6and IL-10 (interleukines 6 or 10). Immunomodulation byL. acidophilus and Bifidobacterium has been observed, inparticular IgA levels and non-specic immunity. Ingestionof probiotic yoghurt has been reported to stimulatecytokine production in blood cells and enhance theactivities of macrophages. Studies showing probioticmicro-organisms and immune system stimulation aresummarized in Table 8.

4. Prebiotics and synbiotics

To maximize effectiveness of bidus products, prebioticsare used in probiotic foods. A prabiotic is a non-digestiblefood that benecially affects the host by selectivelystimulating the growth and/or activity of one or a limitednumber of bacteria in the colon (Shah, 2004, 2006b).Some oligosaccharides, due to their chemical structure,

are resistant to digestive enzymes and therefore pass intothe large intestine where they become available for

L. acidophilus; Lc. lactis ssp. cremoris, Binding of mutagens.

B. lactis; L. gasseri Released bioactive hydrox

showed anticarcinogenic p

L. acidophilus Decreased polyps, adenom

L. acidophilus 145; B. longum 913 Reduction of the risk of c

DNA damage in animals.References

ased diet showed a lower incidence of colon

rimental period, implying that the NCFM

latency period for colon cancer in rats.

along with antibiotics also decreased colon

ad a signicantly lower level of free amines in

Goldin and Gorbach

(1980, 1984a, c).

ily consumption of milk containing NCFM

eduction in the activity of faecal enzymes, X -tase, and azoreductase.

Goldin and Gorbach

(1984b, c)

tics led to anticarcinogenic effects, through

ns in the gut of rats.

Wollowski et al.

(2001a, b)

olic, immunologic, and protective functions in Kasper (1996)

olon cells (antigenotoxicity). Pool-Zobel et al. (1993,

1996); Wollowski

(1998); Ji (1997)

ctivity decreased: b-glucuronidase,se and detoxifying enzyme activity increased.

Benno and Mitsuoka

(1992); Bouhnik et al.

(1996)

ournal 17 (2007) 12621277fermentation by saccharolytic bacteria. Compounds thatare either partially degraded or not degraded by the hostand are preferentially utilized by Bifidobacterium as acarbon and energy sources are referred to as bidogenicfactors. Some bidogenic factors that are of commercialsignicance include fructo-oligosaccharides, lactose deri-vatives such as lactulose, lactitol, galacto-oligosaccharides,and soyabean oligosaccharides. Resistant starch and non-starch oligosaccharides are classied as colonic foods, butnot as prebiotics, because they are not metabolized bycertain benecial bacteria (Shah, 2004).The products that contain both prebiotics and probiotics

are referred to as synbiotics. Synbiotics are a combinationof the effects of probiotics and prebiotics to produce healthenhancing functional food ingredients. Japan is the worldleader in probiotic and prebiotic products. The majority ofyoghurts marketed in Australia, USA and Europe in recentyears contain one or more species of probiotic bacteria andsome form of prebiotics (Shah, 2000a).

5. Alternate products for incorporating probiotics

To realize health benets, probiotic bacteria must beviable and must be available in a high concentration,

Orrhage et al. (1994);

Morotomi and Mutai

(1986)

ycinnamic acids in the human colon which

roperties both in vitro and in animal models.

Couteau, et al. (2001)

as and colon cancer in experimental animals. Gorbach et al. (1987)

olon cancer by inhibiting carcinogen-induced Moschner et al. (2004)

ARTICLE IN PRESS

l le

l as

of

men

iry JTable 5

Probiotic microorganisms and reduction in serum cholesterol

Microorganisms Probiotic function

L. acidophilus Lowered serum cholestero

L. acidophilus; L. casei ASCC 1520, ASCC

1521, ASCC 292, ATCC 15820, and

L. acidophilus ATCC 4962

Highest in vitro cholestero

cholesterol in the presence

media.

Human strain of Ent. faecium 29 men receiving milk fer

N.P. Shah / International Datypically 106 cfu g1 of a product. Despite the importanceof viability, studies have shown low populations ofprobiotic bacteria in probiotic foods (Anon, 1992; Shah,Lankaputhra, Britz, & Kyle, 1995; Shah, Ali, & Ravula,2000). It is questionable whether such products can providethe claimed benets if the populations of probiotic bacteriaare low. Yoghurt is considered the most important carrierof probiotic bacteria. However, a number of factors affectthe loss of viability of probiotic organisms in yoghurt,including acidity of products, acid produced duringrefrigerated storage (also known as post-acidication),

(1081011 cfuL1) led to a dec0.370.41mmolL1 after 6wkno effect.

Enterococcus group of 87 men

decrease in serum LDL conce

than the placebo-group at we

Ent. faecium (Gaio) Was found to be effective in r

lipoprotein cholesterol by 4%

control group.

L. casei TMC 0409 In a single blind parallel stud

(200mLday1) showed signicholesterol, compared with th

supplementation. The levels of

in subjects receiving the ferme

Str. thermophilus TMC 1543 In a single blind parallel stud

(200mLday1) showed signicholesterol, compared with th

supplementation. The levels of

in subjects receiving the ferme

B. longum 913; L. acidophilus 145 Consumption of 300 g yoghur

seven weeks increased high de

0.3mmolL1 (p 0.002) andlipoprotein from 3.24 to 2.48

L. gasseri SBT0270, SBT0274 Exerted hypocholesterolaemic

through deconjugation of bile

L. reuteri CRL 1098 (104 cells d1) Caused a 40% reduction in trhigh density lipoprotein to lo

translocation of the native mi

Albino mice.

B. longum BL1 Led to a signicant lowering

cholesterol, low-density lipop

products compared with the c

lipoprotein cholesterol concen

L. brevis NR1C1684; Ent. faecalis Assimilated more cholesterol (

in the media with 0.2 and 0.4References

vels. Ouwehand et al. (2002)

similation of more than 25mgmL1

cholic acid, compared to other different bile

Liong and Shah (2005)

ted with a human strain of E. faecium Agerbaek, Gerdes, and

ournal 17 (2007) 12621277 1269level of oxygen in products and oxygen permeationthrough the package, sensitivity to antimicrobial sub-stances produced by starter bacteria and lack of nutrientsin milk (Dave & Shah, 1997a, b; Lankaputhra & Shah,1995, 1997; Shah, 2000a; Tamime, Saarela, Sandergaard,Mistry, & Shah, 2005).A number of food products including probiotic yoghurt,

ice cream, frozen fermented dairy deserts, and freeze-driedyoghurt (Capela, Hay, & Shah, 2006) have been employedas alternate delivery vehicles for probiotics. But, due to thepresence of high oxygen content (Lankaputhra & Shah,

rease in serum cholesterol by

, whereas consumption of the placebo had

Richelsen (1995)

and women had a signicantly larger

ntrations decreased throughout the study,

eks 4 and 12 (Po0.05).

Richelsen et al. (1996)

educing both total and low density

and 5%, respectively, compared with the

Agerholm-Larsen, Bell,

Grunwald, and Astrup

(2000)

y, subjects consuming fermented milk

cant increases of high density lipoprotein

e pre-intervention levels after four weeks

triglycerides were also reduced signicantly

nted milk.

Kawase et al. (1999)

y, subjects consuming fermented milk

cant increases of high density lipoprotein

e pre-intervention levels after eight weeks

triglycerides were also reduced signicantly

nted milk.

Kawase et al. (1999)

t supplemented with the 2 microrganisms for

nsity lipoprotein concentration by

decrease ratio of low to high density

(p 0.001).

Kiebling et al. (2002)

effect in rats fed a diet high in cholesterol

salts.

Usman and Hosono

(2000)

iglycerides and a 20 increase in the ratio of

w density lipoprotein without bacterial

croora into the spleen and liver of Swiss

Taranto et al. (1998)

of the serum concentrations of total

rotein cholesterol, and triglycerides in milk

ontrol, while no change in high-density

tration was observed.

Xiao et al. (2003)

in vivo) than the average of the other strains

% (wt/vol) oxgall, respectively.

Pereira and Gibson

(2002)

ARTICLE IN PRESS

H.

n 3

am

hibi

10

he i

ntib

ss t

han

tra

ry JTable 6

Probiotic microorganisms and Helicobacter pylori infection

Microorganisms Probiotic function

L. salivarius Suppressed and eradicated

by lactic acid secretion.

L. gasseri OLL2716(LG21) Signicant improvement o

reduced gastric mucosal in

Bac. subtilis 3 Inhibited H. pylori, the in

pylori isolates ranged from

Bac. clausii Bacteriotherapy reduced t

related to anti-H. pylori a

L. rhamnosus GG, L. rhamnosus LC705;

B. breve Bb99; Propionibacterium

freudenreichii ssp. shermanii JS

Probiotic group showed le

the total symptom score c

eradication therapy in con

N.P. Shah / International Dai12701997) in ice cream type products and injury due to freezingand freeze drying, many of the above mentioned productshave failed to successfully deliver the required level ofviable cells of probiotics. Cheese-based dips could be adelivery vehicle for probiotic bacteria because of its stablepH, the buffering capacity of ingredients used and thepresence of prebiotics (Tharmaraj & Shah, 2004).Other foods such as Cheddar cheese have also been

studied as carriers of probiotic microorganisms. Cheeseshave a number of advantages over fresh fermentedproducts such as yoghurt as a delivery system for viableprobiotic to gastrointestinal tract as they tend to havehigher pH, more solid consistency and relatively higher fatcontent. These offer protection to probiotic bacteria duringstorage and passage through the gastrointestinal tract.Cheeses also have higher buffering capacity than yoghurt.Cheddar cheeses, however, have long ripening time hence

Ent. faecium; Bac. subtilis; Bifidobacterium Produced heat-stable proteina

growth of both antibiotic-resi

L. acidophilus LA5+B. lactis Bb12 Increased eradication ofH. py

with combined use of certain

L. reuteri ATCC 55730 (108CFU) Probiotic supplemented childr

score during eradication thera

L. acidophilus strain LB (lyophilized) 1-week standard therapy incr

group of 120 patients.

L. casei strain Shirota There was a trend towards su

patients.

L. johnsonii La1 There was reduced density ofH

activity among 52 patients.

L. acidophilus NAS Eradication of H. pylori in six

L. casei subsp. rhamnosus (GG); S.

cerevisiae Boulardii; L. acidophilus; B. lactis

Probiotic preparations supple

were associated with lower inc

better treatment tolerability c

L. reuteri strains (JCM1112, JCM1081,

JCM1084 JCM2762, JCM2763 and

JCM2764)

Inhibition by selected L. reute

early stage of colonization inH

strains sharing glycolipid spec

probiotics.References

pylori in tissue cultures and animal models Aiba et al. (1998)

1 subjects infected with H. pylori and

mation following LG21 treatment.

Sakamoto et al. (2001)

tory zone for each of the 21 strains of H.

to 16mm.

Pinchuk et al. (2001)

ncidence of the most common side-effects

iotic therapy compared with placebo.

Nista et al. (2004)

reatment-related symptoms as measured by

ge (P 0.038) throughout the H. pylorist to the placebo group.

Myllyluoma et al. (2005)

ournal 17 (2007) 12621277development of probiotic Cheddar cheese requires a carefulexamination of the suitability of particular strain(s) tomaintain viability throughout the ripening and shelf life(Ong, Henriksson, & Shah, 2006). Two Cheddar cheesescontaining the probiotic DR20

TM

were launched by Main-land (Fonterra) in Australia in 2002 under the name InnerBalance.Another product, which has received much attention, is

soy based isoavone phytoestrogens, found abundantly insoybeans, are an isomeric family of di-phenolic compoundswith structural and functional similarities to humanestrogens. Soybeans and non-fermented soy foods (includ-ing soy extract) contain 8095% of their isoavones asglucoside conjugates, which are biologically inactive andnon-bioavailable (Tsangalis, Ashton, McGill, & Shah,2002). The biologically active (estrogen-like) and bioavail-able aglycone forms of daidzein, genistein and glycitein are

ceous compounds capable of inhibiting the

stant and -sensitive strains of H. pylori.

Tsai et al. (2004); Pinchuk

et al. (2001); Collado et

al. (2005)

lori among 160 dyspeptic subjects in 4 weeks

antibiotics.

Sheu et al. (2002)

en had a signicant reduction of GSRS

py compared to those receiving placebo.

Lionetti et al. (2006)

eased eradication rate of H. pylori in active Canducci et al. (2000)

ppressive effect in an active group of 20 Cats et al. (2003)

. pylori, reduced inammation and gastritis Felley et al. (2001)

of 14 patients after 8 weeks of treatment. Mrda et al. (1998)

menting a standard antiH. pylori regimen

idence of self-reported side effects and with

ompared to placebo.

Cremonini et al. (2002)

ri strains helped to prevent infection in an

. pylori leading to a proposal that L. reuteri

icity with H. pylori have a potential as

Mukai et al. (2002)

ARTICLE IN PRESS

a fr

atie

ma

teria

itab

l sy

roen

arrh

oxy

ciat

fun

e.

epis

he 2

e we

diar

se.

iry JTable 7

Probiotic microorganisms and inammatory bowel disease

Microorganisms Probiotic function

Ent. faecium Decreased duration of acute diarrhoe

L. acidophilus Signicant decrease of diarrhoea in p

L. plantarum Especially effective in reducing inam

enterocolitis in rats, small bowel bac

Reduced pain and constipation of irr

atulence, and pain in irritable bowe

L. reuteri Shortened the duration of acute gast

Shortened acute diarrhoea.

S. cerevisiae Boulardii (yeast) Reduced recurrence of Cl. difficile di

Effects on Cl. difficile and Klebsiella

shortened duration of antibiotic-asso

acute gastroenteritis. Decreased only

symptoms of irritable bowel syndrom

B. bifidum; Str. thermophilus Reduction of rotavirus shedding and

Ent. faecium PR88 Symptomatic improvement in 19 of t

caused by food intolerance for twelv

weight.

S. cerevisiae Boulardii; L.

rhamnosus GG

Helped prevent antibiotic-associated

appeared useful for Cl. difficile disea

N.P. Shah / International Dalinked to the prevention and potential treatment ofhormone-dependent disorders, including osteoporosis,cardiovascular disease and cancer (Tsangalis, Ashton,Wilcox, Shah, & Stojanovska, 2005). Recent research hasshown that isoavone aglycones are absorbed faster and inhigher amounts in humans than their respective glucosides.Intestinal microora, consisting predominantly of Bifido-bacterium, are of signicant importance in hydrolysingingested isoavone glucosides into bioavailable, bioactiveaglycones (Tsangalis et al., 2005), as well as in convertingdaidzein to the biologically potent equol. Fermentation ofplain soy extract with Bifidobacterium sp. is reported tohydrolyse the isoavone glucosides into bioactive agly-cones, increasing the concentration of aglycones from lessthan 10% to approximately 50% of the total isoavonecontent (Tsangalis et al., 2002).

5.1. Products based on bioactive peptides released from milk

proteins by proteolytic probiotics

The functionality of dairy proteins is further enhancedupon liberation of bioactive peptides by proteolysis(Gobbetti, Stepaniek, De Angelis, Corsetti, & Di Cagno,2002). Proteolysis is performed by naturally occurringenzymes in milk, starter cultures and the enzymes of thedigestive tract, resulting in release of many peptides withdifferent biogenic activities such as opioid, hypotensive,immunomodulating, antithrombotic and antimicrobialactivities.References

om gastroenteritis. Marteau et al. (2001)

nts receiving pelvic irradiation. Marteau et al. (2001)

tion in inammatory bowel; e.g.,

l overgrowth in children, pouchitis.

Schultz and Sartor (2000);

Vanderhoof (2000)

le bowel syndrome. Reduced bloating,

ndrome in controlled trial.

Nobaek et al. (2000)

teritis. Marteau et al. (2001)

Shornikova et al. (1997a,

1997b)

oea. Pochapin (2000)

toca resulted in decreased risk and/or

ed diarrhoea. Shortened the duration of

ctional diarrhoea, but not any other

Marteau et al. (2001)

odes of diarrhoea in children in hospital. Saavedra et al. (1994)

8 patients with high volume diarrhoea

eks and a signicant decrease in faecal

Hunter et al. (1996)

rhoea (AAD). S. cerevisiae Boulardii McFarland (2006)

ournal 17 (2007) 12621277 1271Dairy cultures used in production of fermented milkproducts have appreciable proteolytic activity enablingtheir rapid growth in milk. During fermentation, milkproteins, namely caseins, undergo a slight proteolyticdegradation resulting in a number of potentially bioactivepeptides (Table 9). Strains of L. helveticus have beenidentied as highly proteolytic and peptidolytic towardscaseins and their derivatives and, if used in fermentations,they can produce products with a range of bioactivitiessuch as opioid and hypotensive activity (Matar, Amiot,Savoie, & Goulet, 1996).Another group of bioactive peptides, termed angiotensin

I-converting enzyme (ACE) inhibitors, have been exten-sively studied due to their hypotensive role (Conlin et al.,2000). Milk proteins contain a number of ACE inhibit-ing peptides encrypted within their primary structures(Donkor, Henriksson, Vasiljevic, & Shah, 2005). Thesepeptides are liberated by proteolytic action of extracellularand intracellular enzymes of LAB proteolytic system.Strains of L. helveticus appeared to be superior in regardto production of ACE inhibitory peptides in comparisonwith other species tested (Ashar & Chand, 2003). Severalcommercial products containing highly proteolytic strainsof L. helveticus have been developed and marketed aspossessing hypotensive activity, including Calpiss andEvoluss. Calpiss sour milk (Calpis Co. Ltd., Tokyo,Japan), is prepared by fermenting milk using mixed culturecontaining L. helveticus CM4 (CP790) and S. cerevisiae.These organisms are responsible for the release of a range

ARTICLE IN PRESS

Table 8

Probiotic microorganisms and immune system stimulation

Microorganisms Probiotic function References

Lactobacillus strains Microbial interference therapythe use of nonpathogenic bacteria to

eliminate pathogens and as an adjunct to antibiotics.

Bengmark (2000)

Improved mucosal immune function, mucin secretion and prevention of

disease.

MacFarlane and

Cummings (2002)

L. acidophilus NCFM Prevented urogenital infection with subsequent exposure to three pathogens

E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa.

Sanders and Klaenhammer

(2001)

L. plantarum Positive effect on immunity in HIV+ children. Walker (2000)

L. rhamnosus Enhanced cellular immunity in healthy adults in controlled trial. Tomioka et al. (1992)

S. cerevisiae Boulardii In HIV associated acute diarrhoea, 56% of patients who were treated with

S.cerevisiae Boulardii had their symptoms resolved compared with only 6% of

placebo treated patients.

Saint Marc et al. (1999)

L. casei sp.; L. fermentum sp. Can induce cytokine responses in human peripheral blood mononuclear cells. Chen et al. (1999);

Miettinen et al. (1996)

L. salivarius strain Produces a large amount of lactic acid and completely inhibits the growth of

H. pylori in a mixed culture, hence suppressing H. pylori, and reducing the H.

pylori-induced inammatory response.

Aiba et al. (1998)

L. acidophilus DDS-1 Has been shown to induce the production of IL-1a and TNF- a. Rangavajhyala et al. (1997)

L. helveticus Able to release peptide compounds that may have important implications for

the modulation of the cellular immune response.

Matar et al. (2001);

Leblanc et al. (2002)

L. acidophilus TMC 0356 Signicantly increased the production of IL-6, IL-10, IL-12, and TNF-a Morita et al. (2002)

L. rhamnosus sp.; L. plantarum or

L. paracasei ssp. paracasei strains

Induced high levels of IL-10 in human blood mononuclear cells, or monocytes

and highest levels of IL-12 produced by L. paracasei subsp. paracasei strains,

implying its suitability to stimulate cell-mediated immunity.

Hessle et al. (1999)

L. casei Shirota Induced a marked increase in the production of IL-12, probably by

macrophages, which in turn stimulated the production of IFN-g.Kato et al. (1999)

Induced the production of several cytokines, such as IFN- g, IL-1b and TNF-a in mice.

Matsuzaki (1998)

B. adolescentis; B. longum Induced signicantly more pro-inammatory cytokine secretion of IL-12 and

TNF-a than did the infant-type bidobacteria, B. bifidum, B. breve, and B.infantis.

He et al. (2002)

L. plantarum 299v In the presence of the proinammatory cytokine TNF-a, exerted a protectiveeffect by downregulating IL-8 secretion in the human HT-29 colonic epithelial

cell line.

Mccracken et al. (2002)

L. casei DN-114 001, DN-114056,

ATCC-334; L. bulgaricus LB-10 strains

Normal colonic specimens have been obtained from Crohns disease (CD)

patients with neoplasm and inamed ileal specimens after being cultured with

either of these microorganisms.

Borruel et al. (2003)

L. johnsonii La1 Increases transforming growth factor beta mRNA in leukocyte-sensitized

Caco-2 cells.

Haller et al. (2000)

L. johnsonii La1; B. bifidum Bb12

strains

In healthy volunteers receiving a fermented milk product supplemented with

either bacteria for 3 weeks, phagocytosis of E. coli sp. by leukocytes isolated

from the blood was enhanced.

Schiffrin et al. (1995, 1997)

L. casei Shirota; B. breve YIT4064

strains

In mice, oral administration of either bacteria activated the humoral immune

system.

Yasui et al. (1999)

L. casei ssp. rhamnosus GG Prevents cytokine-induced apoptosis in intestinal epithelial cell lines in culture

by activating the anti-apoptotic Akt/protein kinase B, and by inhibiting the

pro-apoptotic p38/mitogen-activated protein kinase by stimulating the

production of TNF-a, IL-1b, IL-1a, or INF-g.

Yan and Polk (2002)

N.P. Shah / International Dairy Journal 17 (2007) 126212771272

lar. Other probiotic organisms including Ent. faecium,

ARTICLE IN PRESS

thei

Leu-Asn-Val-Pro-Gly- L. delbrueckii ssp.

iry JS. cerevisiae Boulardii and Propionibacterium have poten-tial to be used in probiotic products. Several health benetshave been claimed for probiotic bacteria. Yoghurt is themost important delivery vehicle for probiotic organisms.of potent bioactive peptides including two tripeptides, Val-Pro-Pro and Ile-Pro-Pro (Nakamura et al., 1995; Vasiljevic& Shah, 2007).

6. Conclusions

Probiotic products containing L. acidophilus, Bifidobac-terium spp. and L. casei are becoming increasingly popu-

glu-Ile-Val-Glu bulgaricus SS1

Asn-Ile-Pro-Pro-Leu- Lc. lactis ssp.

Thr-Glu-Thr-Pro-Val cremoris FT4

aAdapted from Vasiljevic and Shah (2007).Table 9

Some examples of the identied bioactive peptides in fermented milk and

Sequence Microbial agent

Val-Pro-Pro L. helveticus CM4 &

Ile-Pro-Pro S. cerevisiae

Val-Pro-Pro L. helveticus

Ile-Pro-Pro LBK16H

Asn-Leu-His-Leu-Pro- L. helveticus NCC

Leu-Pro-Leu-Leu 2765

Tyr-Pro-Phe-Pro-Glu- L. helveticus NCC

Pro-Ile-Pro-Asn 2765

Tyr-Pro L. helveticus CPN4

N.P. Shah / International DaCheddar cheese, dips and spreads are becoming popular asalternate products for incorporation of probiotics. Proteo-lytic strains of probiotic bacteria are used to releasebioactive peptides such as ACE-inhibitor peptides forfurther improving the health benets of probiotic foods.

References

Agerbaek, M., Gerdes, L. U., & Richelsen, B. (1995). Hypocholester-

olaemic effect of a new fermented milk product in healthy middle-aged

men. European Journal of Clinical Nutrition, 49, 346352.

Agerholm-Larsen, L., Bell, M. L., Grunwald, G. K., & Astrup, A. (2000).

The effect of probiotic milk product on plasma cholesterol: A meta-

analysis of short-term intervention studies. European Journal of

Clinical Nutrition, 54, 856860.

Aiba, Y., Suzuki, N., Kabir, A. M., Takagi, A., & Koga, Y. (1998). Lactic

acid-mediated suppression of Helicobacter pylori by the oral admin-

istration of Lactobacillus salivarius as a probiotic in a gnotobiotic

murine model. American Journal of Gastroenterology, 93, 20972101.

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