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Probiotic lactic acid vs. bacilli: pros and cons ARTHUR C. OUWEHAND1*, SOFIA FORSSTEN1, MARKUS LEHTINEN1, ELIZABETH GALBRAITH2, ELLEN DAVIS2 *Corresponding author 1. Active Nutrition, DuPont Nutrition & Health, Sokeritehtaantie 20, 02460 Kantvik, Finland Arthur C. Ouwehand 2. Animal and Environmental Applications, DuPont Nutrition & Health, W227 N752 Westmound Drive, Waukesha, WI 53186, USA

KEYWORDS: probiotic, , , Bifidobacterium, , spores

ABSTRACT: Both lactic acid bacteria (LAB) and bacilli are commercialised as probiotics; LAB are mainly utilised as live and active or dormant cells, while bacilli are utilised as spores. Belonging to very different genera, they have different technological properties. LAB have a longer history of use as probiotics and therefore have a broader base as far as health efficacy documentation is concerned. The question therefore sometimes arises; strains from which of these groups make better probiotics? Considering the fact that probiotics, by definition, have to be viable in sufficient numbers and have to have documented health benefits, this is not a relevant question. Any microbial strain has to fulfil these criteria in order to qualify as a probiotic. The current paper reviews and compares the available data for both groups of organisms. Pre - Probiotics

INTRODUCTION In order to supply consumers with products that provide meaningful levels of probiotics, several requirements have There is no legal description of probiotics. However, the most to be fulfilled by probiotic strains selected for industrial commonly accepted definition is from a FAO/WHO working production. Quality-control must be vigilantly performed, group: “Live microorganisms which when administered in assuring identity, viability and absence of contaminants. adequate amounts confer a health benefit on the host”. This Other properties, that are related to the physiological state implies that probiotics must be alive in sufficient number at of the strain, such as adhesion; bile and acid resistance; the time of use and must have documented health benefits. effects on carbohydrate, protein, and fat utilisation; and, The most commonly used probiotics for human use are especially, colonisation properties (usually as faecal strains that belong to the genera Lactobacillus and recovery) and immunogenicity (e.g. in vitro as cytokine Bifidobacterium, although the latter are genetically distinct induction in peripheral blood mononuclear cells) may also from the former, for convenience they will be grouped need to be assessed (1). Long-term industrial processing and here as lactic acid bacteria (LAB). An emerging group of storage conditions may influence probiotic properties. Thus, probiotics are strains that belong to the Bacillus. An in addition to technological properties, functional properties often arising question is which one makes a ‘better’ probiotic should be considered in quality-control measures. as both groups have pros and cons. The present review compares the characteristics for both probiotic groups. The production of probiotics follows a general process LACTIC ACID BACTERIA consisting of several steps, starting with fermentation. Ingredient choice is a crucial step in the fermentation process; final product form and allergen potential are Lactic acid bacteria (LAB) are Gram-positive organisms factors that must be considered. Growth conditions exhibiting a DNA G+C content of less than 50 %. have to be optimised with the right oxygen tension, pH, Phylogenetically they belong to the -Bacillus and temperature. For organisms belonging to the genus subdivision of Gram-positive eubacteria. Lactobacilli are Bacillus, induction of spores is promoted near the end of the facultative anaerobes. The genus Bifidobacterium belongs fermentation process, typically through nutrient depletion. to the Actinomycetes subdivision of the Gram-positive After fermentation, the large biomass of probiotics produced is concentrated either by centrifugation or membrane filtration. As spore formers, bacilli are more robust and resist the downstream processing better then LAB, and therefore traditional spray drying is commonly utilised in their production. To preserve LAB viability, maintaining biological membrane integrity and associated proteins through the drying process, is critical in order to maximise cell recovery following rehydration. Cryoprotectants, such as e.g. lactose or sucrose, are used to stabilise the membrane and to minimise the degrading effects caused during the freezing and drying steps. The stabilised material can then be frozen for the freeze-drying process. Formation of ice crystals is minimised during freeze- drying. Solid water is removed via sublimation. The chosen residual moisture content and the stabiliser components have an important impact upon shelf-life. For both LAB and bacilli, the dry material is milled to appropriate particle size, 30-100 µm, for blending and packaging.

13 eubacteria and has a high G+C content of approximately but-non-culturable state, with 60%. Although bifidobacteria are taxonomically distant possible beneficial effects from LAB, they are commonly grouped together as they all such as immune modulation produce lactic acid as a major metabolic end product and and carcinogen binding share habitats such as the intestinal tract and dairy. For the in the host (3). Thus, for purposes of this review, we will consider bifidobacteria to be some probiotics it might part of the LAB group. be sufficient that they grow well during the initial Application in food production steps in order LAB have traditionally been used in the fermentation of to obtain high enough various foods. Since the beginning of the 20th century, they numbers in the product, have also been recognised as providers of health benefits while good viability during and are included for this purpose primarily in fermented storage and/or passage dairy products and dietary supplements. In foods, LAB will through the GI tract may not usually be present as active organisms; limiting their shelf be required. life to about a month. In dietary supplements, however, the microbes will be in a dormant desiccated state expanding Health effects their shelf life to up to 24 months, provided a low water Sequencing technologies have activity (Aw<0.20) is maintained. Application of probiotics increased our understanding of the role in food and feed is limited by water activity, temperature of microorganisms on health and disease. and pH and to a lesser extend oxygen. Of course, all these Function and composition of the gut microbiota may factors combined and the time they are exposed to stressful be modulated by introducing beneficial bacteria, opening conditions influences the level of live microbes, Figure 1. intriguing possibilities for improving health.

Gastrointestinal health benefits The impact of LAB has been studied extensively on prevention and treatment of gastrointestinal diseases. Irritable bowel syndrome (IBS) manifests itself as a combination of functional gut problems, like diarrhoea, constipation, intestinal pain, and bloating. It has been estimated that ~10% of the western population have

Pre - Probiotics indications of IBS. The underlying cause of IBS is still unclear, but a meta-analysis has shown that LAB consumption improves symptoms of IBS in a species specific manner (4). Infectious diarrhoea is a global problem and frequent cause of mortality especially in children. Diarrhoea may also be caused by outgrowth of opportunistic pathogens in the gut or when antibiotic treatment disturbs the balance of the microbiota. A recent meta-analyses has shown convincingly that prophylactic consumption of LAB reduce the risk of antibiotic associated diarrhoea, Clostridium difficile Figure 1. Schematic representation of the effect of temperature and water activity (Aw) associated diarrhoea (5), infectious diarrhoea (6), and on viability of LAB probiotics necrotising enterocolitis (7). Effects of LAB consumption in the treatment of chronic diarrhoea are not yet conclusive but indications of some benefit in children have been found (8). Feed applications with long shelf lives at ambient Inflammatory bowel disease (IBD) is a condition vol 24(6) - November/December 2013 -

temperatures, relatively high processing temperatures characterised by dysbalance in gut microbiota and and high water activity are especially challenging for LAB impaired recognition of gut microbiota by the immune formulations. Combination of LAB with prebiotics to create system, and IBD occurs in two forms: Crohn’s disease and a synbiotic is sometimes suggested but there is currently ulcerative colitis (UC). LAB consumption may reduce the insufficient data available to substantiate a performance relapse rate in UC but the evidence is far from clear (9). benefit with such combination. Also the effect of LAB on Crohn’s disease is still inconclusive and it has been suggested that LAB could potentially Viability/activity (in product and intestine) increase relapse rate (10). In the light of the current research FOOD Industry Hi Tech Probiotic strains must retain their characteristics and viability on Crohn’s disease it is not surprising that consumption during the production processes and storage in different of supplemental LAB probiotics may not be effective in Agro surroundings as well as transit through the GI tract. For ameliorating symptoms. Crohn’s disease has a genetic products where the probiotic is actively metabolising, as is component that presents itself as dysfunctional recognition the case in LAB fermented dairy products, the stability can of commensal bacteria by the mucosal immune system. be a function of the inherent capabilities of the bacteria as Thus, increasing bacterial load in the small intestine may not well as the physical properties of the food matrix. The matrix be beneficial. and type of product has a large impact on the viability and shelf life (2). If stability is defined as the ability to survive Immune system modulation under given circumstances, it cannot be firmly linked to Humans are born sterile and it has been shown that the characteristics of a particular genus or species, but is colonisation of the intestine by microbiota from the dependent on the particular strain of LAB and the culture environment (vagina, skin, breast milk) is required for normal conditions used. Viability is not easily defined in microbes. development of mucosal immune system. The signalling Culturable probiotics are beyond doubt viable. However, between the host and gut microbiota results in homeostasis microbes can be damaged and enter a so-called viable- that may be perturbed by changes in microbiota (or host).

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, . A , spores spores -containing probiotics are probiotics are B. coagulans Bacillus are utilised as are utilised , and Bacillus have been mislabeled, when they have been shown species to form a protective spore species to form a protective spore species (19). This mislabelling may spores to various stressful conditions. spores to various stressful B. subtilis contains a large and diverse array large and diverse array contains a Bacillus Bacillus , B. anthracis and certain strains of B. cereus and certain strains B. anthracis Bacillus has a long history of human consumption in the has a long history of human consumption in the bacteria, particularly of the genus of the particularly bacteria, feed applications. and animal human for both probiotics The genus range of environments making possible the of species, the capacity. With and their wide metabolic they inhabit of exception However, of Bacillus are non-pathogenic. most species food surgery rooms and cause problems in e.g. they may due to the resistance of their spores to processing equipment desiccation, Table 1. disinfectants, heat and dietary Species most commonly found in commercial supplements include , Bacillus claussii Bacillus recent review (19) provides a sampling of range of health- products marketed for human use and the many probiotic promoting functions claimed. Historically, products containing spore-formers often as containing to contain other phenotypic have been the result of classification using be hard to or biochemical means which can sometimes and can yield standardise, are subject to interpretation, growth conditions. variable results depending on laboratory Also B. coagulans is sometimes still incorrectly referred to Lactobacillus sporogenes. Current classification is typically rRNA gene based on molecular methods such as 16S is not without sequence comparison, however this method are not drawbacks, as well-defined species boundaries rDNA can prevent always clear-cut and divergence in 16S (20). the resolution of strain and species relationships Application in food and feed capability of Bacillus The usually included in the form of prophylactics to protect usually included in the form of prophylactics to protect against gastrointestinal disorders such as diarrhea in children or as health food and dietary supplements for the promotion of general health and well-being. In addition to the supplement and health food market, a product has been shown to be a good candidate as an addition to baking products due to its ability to maintain viability in both the raw baking product as well as the final product after the baking process (21). Furthermore, B. subtillis form of natto; a traditional Japanese product consisting of fermented soy beans. Combination of with prebiotics to generate a synbiotic is not commonly proposed. Viability/activity in product and intestine Spore-forming bacteria are ubiquitous in nature: found in air, soil, water, and many extreme environments. Bacterial endospores have evolved to survive nutrient deficient conditions which are not conducive for viability of vegetative cells (22). The spore form is dehydrated and consists of a core containing a condensed chromosome, a peptidoglycan-rich cortex layer and one or more layers of proteinaceous material referred to as the spore coat (23). coat makes this bacterium relatively stable in a variety of coat makes this bacterium relatively stable in a variety of food and animal feed applications. Bacillus

This resistance is of course strain dependent and is influenced by the duration of strain dependent and is influenced by the This resistance is of course acid bacteria'; 'Aw=water activity' the exposure. 'LAB=lactic Table 1. Resistance of LAB and Table 1. Resistance of LAB Clostridium-Bacillus Common cold is typicallyCommon cold viruses that arecaused by the immunecleared by signalling from microbiota.signalling from system. haveMeta-analyses immune response by changingimmune response ofshown that consumption LAB reduces common coldLAB reduces common infection rate and duration of the opportunities to modulate host modulate to opportunities symptom episodes (11). Immune responses are beneficial when targeted This interaction provides unique provides This interaction Lactobacillus or Bifidobacterium specifically against a pathogen, but can be detrimental if uncontrolled ordetrimental if uncontrolled nonspecific in nature, during annonspecific in nature, allergic reaction for instance. Some LABallergic reaction for instance. strains have shown anti-inflammatory potential that mightstrains have shown anti-inflammatory potential subdivision of Gram-positive eubacteria. Spore-forming Taxonomy Bacilli are aerobes or facultative anaerobes and belong, similar as the genus Lactobacillus, to the SPORE-FORMING BACTERIA AS PROBIOTICS Regulatory/safety The strains of LAB probiotics commonly used today belong to species that in Europe have the status of Qualified Presumption of Safety (QPS) (18). In the USA, many strains have self-approved or Food and Drug Agency (FDA) approved GRAS (generally recognised as safe) status for use in dietary supplements or fermented foods. Although sporadic case reports of infection exist, the general perception is that strains from these species are safe. Severely immune compromised subjects may be at risk for infection by strains from these species (and most other microbes for that matter); their use should happen under medical supervision. Improved metabolism and Prevalence of life-style diseases, like type-2-diabetes globally. metabolic syndrome, has steadily increased harvest, The microbiota is closely linked to e.g. energy bile-acids. detoxifying compounds and de-conjugating to lower totalConsumption of LAB was shown in a meta-analysis suggestalso study clinical recent a and (14), cholesterol LDL and (15). a role for preventing obesity and diabetes Conclusively in these studies indicate a role for LAB consumption preventing life-style diseases. that is The liver is a central metabolic organ in humans coming the first organ to handle most of the metabolites the liver is from the intestine. In hepatic encephalopathy the intestine. unable to detoxify substances arising from hepatic LAB consumption has been shown to improve encephalopathy (16) that is likely linked to improving detoxification or intestinal barrier. A recent clinical study also suggests a possible role for LAB consumption in the treatment of non-alcoholic fatty liver disease (17). reduce symptoms of allergy and accordingly some beneficialreduce symptoms of allergy and accordingly of seasonaleffects have been found in reducing severity seemsallergy symptoms (12). In addition, LAB consumption severity of atopicto be beneficial against development and if probiotic use isdermatitis and eczema in children especially started during pregnancy (13). 16

Agro FOOD Industry Hi Tech - vol 24(6) - November/December 2013 Pre - Probiotics of mice andpigshavereportedthat the administration more resistanttopathogenicbacteria(19).Studiesin gastrointestinal microbialcommunityandmakingit a competitiveexclusionresponsebyalteringthe produce antimicrobialcompoundsthatprovide attributed tothepropensityoftheseorganisms The probiotichealthbenefitsof Health effects through theirconsumptioninfood(25). numbers inhumanfecesthancanbepresumed gastrointestinal tract(24)andarefoundathigher spores germinate withintheenvironmentof growing numberofanimalstudiessuggestthat the intestineofinsects,animalsandhumans(24)a (19). SporesofBacillus proliferating andfinallyre-sporulatingpriortodefecation appropriate conditionsforgerminationintheintestine, environments, presentinasporestateuntilreaching perhaps evolvedtoexistinsymbiosiswithingastrointestinal Bacillus microorganisms, currentprevailingtheoryproposesthat species havetraditionallybeenconsideredsoil-borne Though spore-formingbacteriasuchas pancreatic enzymeconditionsfoundintheintestine(24). gastric conditionsinadditiontothehigherbileand demonstrated that spore-forming bacteriaattractiveprobiotics.Ithasbeen Their abilitytosurvivethegastricbarrieralsomakes shelf-life. accurate probioticdosesoveranextendedproduct little effectonviability,helpingtoensurethedeliveryof storage atroomtemperatureinadesiccatedformhas through arangeofmanufacturingprocesses.Inaddition, to productioninavarietyofdryorliquidformatsand These propertiesmakespore-formingprobioticsfavourable high heatpelletingconditionsinanimalfeedapplications. shelf lifestabilityindryfoodmatricesandresistancetothe high temperaturesupto80-85°C(22),therebyproviding such asUVradiation,freeradicalandenzymeactivity, The sporecoatprotectstheorganismfromharshconditions bacteria intermsofviabilityduringproductionandstorage. advantage toendospore-formingbacteriaoverlacticacid As probiotics,theheartysporecoatconveysadistinct , and perfringens as disease, aswell with human associated C. difficile pylori, and Helicobacter jejuni, Camphylobacter flexneri aureus, bacteria including potentially pathogenic against anumberof to haveinhibitoryactivity probiotics havebeenreported the gastrointestinaltract. for beneficialmicroorganismsin more hospitablemicroenvironment ingestion of in thegastrointestinaltract(26),indicating Bacillus Clostridium , cannotonlysurvive,butcanthriveandhave Shigella enhancedpopulationsof

, Bacillus C. difficile, Bacillus sporesresultsina specieshavebeenisolatedfrom Bacillus speciessurvivewellunder Bacillus

Lactobacillus Bacillus aremainly

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other strainsof by humanperipheralbloodmononuclearcells(29),and signified bytheinductionofinterleukin(IL)-10production subtilis as indicatedbyapparentlyconflictingreportsofa specific totheparticularstrainandimmunecellevaluated, typical forprobiotics,theeffectsof Caco-2 cellswhenactivatedbyB.subtilis produced byepithelial-likehumancoloncarcinoma factor kappa-β(NF-kB)activationwerereportedtobe Several cytokinesassociatedwithnucleartranscription Bacillus ingestion (27),indicatingadirectinteractionbetween mesenteric lymphnodesoftheintestinaltractafteroral were foundtobepresentwithinPeyer’sPatchesand tract andspecificimmunecells(19).B.subtilisspores the lymphoidtissuesassociatedwithgastrointestinal health benefitisthroughimmunomodulatoryeffectson Another importantmechanismbywhich Salmonella entericainanimals(26). placebo group(30).Innatto, consuming of rheumatoidarthritisreportedreducedpainwhen associated diarrheawithsomesuccess(5).Sufferers report theeffectof Human studies,however,arescarce(19).Twostudies production whenexposedtomacrophagecultures(27). an inflammatoryresponsethroughtheinductionofIL-6 probioticelicitingananti-inflammatoryeffect probioticsandthegastrointestinalimmunesystem. B. coagulansGBI-30,6086ascomparedtothe benefit bytheproductionofvitaminK2,but B. subtilis Bacillus this isnotaprobioticbenefit. Regulatory andfafety andB.pumilussporesresultingin Members ofthe with knownpathogenicityinclude B. anthracisandcereus, associated withB.cereus, to sixenterotoxinscommonly caused bytheproductionofup (31). Thediseasesyndromeis type oremetic-typedisease spores canproduceadiarrhea- contaminant andingestionof B. cereusisafoodspoilage B. cereusisstraindependent. although thepathogenicityof therefore theabsenceof probioticsonantibiotic- genes associatedwiththese enterotoxins isoftenusedasa screen for B. subtillis as otherpotential probiotics. Fewvirulence factors havebeenreported in other some havedemonstrated commonly used as commonly usedas The strainsof cell culturelines(31). cytotoxic effectsinhuman invades andproduces whether ornotastrain conducted byassessing for virulenceistypically have the status of Qualified have thestatusofQualified to speciesthatinEurope probiotics todaybelong result anadditionalscreen cytotoxic effects.Asa by the European Food by theEuropean Food Presumption of Safety (QPS) Bacillus providesahealth Bacillus B. cereusaswell Bacillus Bacillus (natto) (28).As seemtobe Bacillus species,but genus exertsa Bacillus B. B.

Safety Authority (EFSA)(18). Based on the possibility of some 4. Ortiz-Lucas, M., A. Tobias, et al., Rev Esp Enferm Dig, 105, Bacillus sp. with QPS status to cause foodborne illness, the 19-36 (2013). absence of food poisoning toxins as well as surfactant and 5. Hempel, S., S.J. Newberry, et al., JAMA, 307, 1959-1969 enterotoxic activities must be demonstrated (18). In the US, (2012). 6. Ritchie, M.L. and T.N. Romanuk, PLoS One, 7, e34938 (2012). the use of Bacillus as probiotics is regulated by the FDA, 7. Alfaleh, K., J. Anabrees, et al., Cochrane Database Syst Rev, and may be granted GRAS status approval on an individual CD005496 (2011). case-by-case basis. 8. Bernaola Aponte, G., C.A. Bada Mancilla, et al., Cochrane Database Syst Rev, CD007401 (2010). 9. Sang, L.X., B. Chang, et al., World J Gastroenterol, 16, 1908- CONCLUSION 1915 (2010). 10. Butterworth, A.D., A.G. Thomas, et al., Cochrane Database By definition, probiotics need to have documented health Syst Rev, CD006634 (2008). benefits. LAB have a substantial volume of documented 11. Hao, Q., Z. Lu, et al., Cochrane Database Syst Rev, CD006895 health benefits in humans, even though to date no (2011). 12. Das, R.R., M. Singh, et al., World Allergy Organ J, 3, 239-244 probiotic strain has received a positive opinion from EFSA (2010). for a health claim in human nutrition. Bacilli currently 13. Pelucchi, C., L. Chatenoud, et al., Epidemiology, 23, 402-414 have a much smaller scientific basis for health benefits in (2012). humans. Although interestingly EFSA has issued positive 14. Guo, Z., X.M. Liu, et al., Nutr Metab Cardiovasc Dis, 21, 844- opinions for selected Bacillus strains in animal nutrition 850 (2011). applications. 15. Kadooka, Y., M. Sato, et al., Eur J Clin Nutr, 64, 636-643 By definition, probiotics also have to be delivered in (2010). sufficient amounts. With their robustness,Bacillus probiotics 16. Holte, K., A. Krag, et al., Hepatol Res, 42, 1008-1015 (2012). 17. Aller, R., D.A. De Luis, et al., Eur Rev Med Pharmacol Sci, 15, can easier provide the required dose to the consumer in Pre - Probiotics almost any format; this is more challenging for LAB, Table 1. 1090-1095 (2011). 18. Hazards, E.P.o.B., EFSA J, 10, 3020 (2012). The bottom line, however, is that both criteria have to be 19. Cutting, S.M., Food Microbiol, 28, 214-220 (2011). fulfilled for each particular strain or strain combination. 20. Maughan, H. and G. Van der Auwera, Infect Genet Evol, 11, Thus there has to be clinical documentation for the 789-797 (2011). particular product; no level of robustness can replace 21. Permpoonpattana, P., H.A. Hong, et al., Benef Microbes, 3, that. At the same time, the product has to deliver the 127-135 (2012). minimum required dose at end of shelf life; clinical results 22. Nicholson, W.L., N. Munakata, et al., Microbiol Mol Biol Rev, are irrelevant if insufficient levels of probiotics are provided. 64, 548-572 (2000). Because these criteria have to be fulfilled for each strain or 23. Henriques, A.O. and C.P. Moran, Jr., Annu Rev Microbiol, 61, combination of strains, the original question what is a better 555-588 (2007). probiotic, LAB or bacilli becomes irrelevant. Organisms from 24. Barbosa, T.M., C.R. Serra, et al., Appl Environ Microbiol. 71, 968-978 (2005). either group have the potential to be good probiotics; but 25. Hong, H.A., E. To, et al., Res Microbiol, 160, 375-379 (2009). they have to comply with the definition. 26. Baker, A.A., E. Davis, et al., Appl Environ Microbiol. 76, 2961- 2967 (2010). 27. Duc le, H., H.A. Hong, et al., Vaccine, 22, 1873-1885 (2004). REFERENCES AND NOTES 28. Hosoi, T., R. Hirose, et al., Int J Food Microbiol, 82, 255-264 (2003). 1. Pineiro, M. and C. Stanton, J. Nutr, 137, 850-853 (2007). 29. Foligne, B., E. Peys, et al., Clin Nutr, 31, 987-994 (2012). 2. Mäkeläinen, H., s. Forssten, et al., Int Dairy J, 19, 675-683 30. Mandel, D.R., K. Eichas, et al., BMC Complement Altern Med, (2009). 10, 1 (2010). 3. Ouwehand, A.C. and S.J. Salminen, Int Dairy J, 8, 749-758 31. Ceuppens, S., N. Boon, et al., FEMS Microbiol Ecol, 84, 433- (1998). 450 (2013).

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