End of Project ReportGrowing  public  awareness  of
diet-related health benefits has fuelled the demand for probiotic foods.
These foods contain probiotic bacteria which are described as live microbial
supplements that improve the intestinal microbial balance and are intended for maintenance of health and/or the prevention of disease. Probiotic bacteria for human
use must be proven to be safe and beneficial, and should preferably be of human origin as
evidence suggests that these bacteria are species specific and perform best in the species
from which they were isolated. They must also retain both viability and efficacy in a
particular food product throughout its shelf-life, and following consumption. Above all
however, probiotic food products must be proved effective in controlled validated clinical
trials.
Dairy foods, including in particular, fermented milks and yogurt are among the best
accepted food carriers for probiotic cultures. The aim of this study was to develop new
probiotic foods, particularly, the production of high quality Cheddar cheese containing high
levels of probiotic bacteria.Department of Agriculture, Food and the Marin

Collins, K.

Fitzgerald, Gerald F

Gardiner, Gillian E.

Ross, R Paul

STANTON, CATHERINE

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DAIRY PRODUCTS RESEARCH CENTREMOOREPARKEND OF PROJECT REPORT 1999:    DPRC No. 29New Probiotic CheddarCheeseDrs. Catherine Stanton and Paul RossCheddar cheeses containing high levels of probiotic strains followingripening, were successfully produced using normal manufacturingprocedures.  The sensory quality of the cheeses was not adverselyaffected by the addition of probiotic strains and flavour was in factenhanced by one probiotic strain. A patent has been filed on theprocess which has attracted intense industry interest. The Science of Farming and Foodfor maintenance of health and/or the prevention of disease. Probiotic bacteria for humanuse must be proven to be safe and beneficial, and should preferably be of human origin asevidence suggests that these bacteria are species specific and perform best in the speciesfrom which they were isolated.  They must also retain both viability and efficacy in aparticular food product throughout its shelf-life, and following consumption.  Above allhowever,  probiotic food products must be proved effective in controlled validated clinicaltrials.Dairy foods, including in particular, fermented milks and yogurt are among the bestaccepted food carriers for probiotic cultures. The aim of this study was to develop newprobiotic foods, particularly, the production of high quality Cheddar cheese containing highlevels of probiotic bacteria. Main Conclusions and Achievements*  Cheddar cheeses containing high levels of probiotic strains following ripening, weresuccessfully produced using normal manufacturing procedures.  The sensory quality of thecheeses was not adversely affected by the addition of probiotic strains and flavour was infact enhanced by one probiotic strain.A patent has been filed on the process which has attracted intense industry interest andcommercial trials are being conducted in one case.*   In pig feeding trials it was shown that mature Cheddar, containing high levels of probiotic bacteria compares favourably with fresh yogurt as an effective delivery system in colonisingthe gastrointestinal tract.A positive immune response (serum IgG) was found in pigs fed the probiotic strains.* Spray dried skim milk powders containing high numbers of probiotic strains, (even afterstorage at 4ºC for 3 months), were successfully produced for use in functional foodsincluding Cheddar cheese, yogurt, ice cream, etc.  Research and Results Selection of Probiotic StrainsA number of probiotic strains (Fig 1), including Lactobacillus and Enterococcus spp., were assessed for use in Cheddar cheese.The probiotic Lactobacillus strains used in this study were obtained from the MicrobiologyDept., University College Cork under a restricted materials transfer agreement.  Thesestrains had previously been isolated from the human gastrointestinal tract, andcharacterised in detail with regard to their probiotic potential at UCC.  In this respect, thesestrains have been shown to be acid and bile tolerant, adherent to human epithelial cells,non-pathogenic and to have desirable antibiogram profiles, in addition to contributing to areduction in disease activity in Crohns disease patients.32Functional Foods in Relation to Health and Disease(New Probiotic Cheddar Cheese)ARMIS No.  4266Project Team:C. StantonP. RossG. Fitzgerald*K. Collins*G. Gardiner*Dept of Microbiology, University College CorkThe Dairy Products Research Centre Moorepark, Fermoy, Co. Cork.This project forms part of a major collaborative research programme intoprobiotic micro-organisms and their application in functional fooddevelopment, between University College, Cork and Teagasc Dairy ProductsResearch Centre, Moorepark.Teagasc acknowledges with gratitude grant aid under the Food Sub-Programme (Sub-Measure 3 (ii) - Institutional R&D) of the OperationalProgramme for Industrial Development.  The Programme is managed by theDepartment of Agriculture and Food supported by EU and national funds.ISBN: 1 84170 122 XDPRC No. 29© Teagasc, September 2000Teagasc 19 Sandymount Avenue EUROPEAN UNIONBallsbridge Dublin 4 European Regional Development FundSummary andConclusionsGrowing public awareness ofdiet-related health benefits has fuelled the demand for probiotic foods.These foods contain probiotic bacteria which are described as live microbialsupplements that improve the intestinal microbial balance and are intendedThe Science of Farming and FoodThe incorporation of these strains did not adversely effect cheese quality (aroma, flavourand texture) while the incorporation of the Enterococcal strain actually improved Cheddarflavour.  This probiotic cheese contained higher levels of odour-active volatile compoundscompared with the control, as quantified by GC-MS head-space analysis, as well asincreased levels of free amino acids, quantified by HPLC.  These Cheddar cheeses can be manufactured to contain high levels of probiotic strains (108cfu/g cheese) at a relatively low cost to the manufacturer using normal make procedures.A patent was filed on the process for the manufacture of probiotic cheese and the projecthas attracted intense industry interest.Industrial trials have been undertaken to examine the feasibility of scale-up to commercialscale while parallel clinical trials have recently been completed to confirm the health-promoting properties of this new functional cheese product.Feeding trialsIn an in vitro model system, Cheddar cheese was found to have a greater protective effectthan yogurt upon exposure of  probiotic, Lb. paracasei, NFBC 338  culture to porcinegastric juice pH 2.In vivo follow-up trials were undertaken to evaluate Cheddar cheese as a food deliveryvehicle for probiotic bacteria by supplementing the diet of pigs with probiotic Cheddar andyogurt.  The probiotic strain - antibiotic (rifampicin) resistant variant of Lb. paracasei NFBC338 was recovered from the porcine small intestine in similar numbers (104 - 105 cfu/ml)whether ingested in Cheddar cheese (total ingested = 108 - 109 cfu) or yogurt (total ingested= 1011 cfu) (Fig 4).5The probiotic strain Enterococcus faecium Fargo 688® was obtained from QuestInternational. In a human clinical trial, it had already been demonstrated that consumptionof this led to alleviation of the symptoms of irritable bowl syndrome in humans (Allen et al.,1996) and so demonstrating potential for use as a probiotic strain.Survival of probiotic strains in Cheddar cheeseA method of distinguishing the added probiotic strains from the normal non-starter lacticacid bacteria (NSLAB) in mature cheese was successfully developed.  It involved the use ofarbitrarily selected oligonucleotides in polymerase chain reactions (PCR) to generatediscrete DNA fingerprints of the strains.  This method was successfully employed foridentification of the probiotic strains following isolation from Cheddar cheese.  In these studies it was demonstrated that two strains of Lb. paracasei grow and sustain high viability in cheese (Fig 2) while Lb. salivarius strains die during the ripening period.  Growthof these strains in milk was found to be relatively slow, but MRS medium was capable ofsupporting the growth of high cell numbers of these strains (~2 x 109 cfu/ml) withcorresponding rapid growth rates.  Enterococcus faecium Fargo 688® was also found to survive to high numbers (4 x 108cfu/g) in Cheddar cheese during ripening at 8ºC for 15 months (Fig 3) and in yogurt (4 x107 cfu/g) during storage at 4ºC for 21 days.  4Survival of Probiotic Lactobacillus paracaseiNFBC 338 in pilot-scale cheese trials98765432100                          50                            100                             150                            200Time (Days)Log CFU/g+NFBC 338+NSLAB starterFig. 2.* porcine origin* porcine feeding studies* human feeding studies* mode of action* alleviation of IBSsymptoms in humans* acid resistant* bile tolerant* adherent* human origin* antimicrobial production(UCC 118,1)* murine feeding studies* IBD alleviation in murine model* human feeding studiesE. faecium PR88(Quest Intl.)Lactobacilli (UCC)Biological PropertiesFig 1.storage of the powders at 4ºC for 2 - 3 months.PublicationsAllen, W.D., Linggood, M.A. and Porter, P. (July 1996).  European patent 0508701B1.Gardiner, G., Ross, R.P., Collins, J.K., Fitzgerald, G.  and Stanton, C.  (1998). Developmentof a probiotic Cheddar cheese containing human-derived Lactobacillus paracasei strains.Applied and Environmental Microbiology, 64, 2192-2199.Stanton, C., Gardiner, G., Lynch, P. B., Collins, J.K., Fitzgerald, G. and Ross, R.P. (1998).Probiotic Cheese. International Dairy Journal, Vol. 8, Issue 5-6, pages 491-496.76In a further feeding trial involving 8 pigs per group, a rifampicin resistant variant of theprobiotic strain Enterococcus faecium Fargo 688® was fed for 21 days at a mean dailyintake of 1.3 x 1010 cfu per day from Cheddar cheese or 3.7 x 109 cfu per day from yogurt.During the feeding period, Cheddar cheese yielded a significantly (P < 0.05) higher meanfaecal probiotic count (2 x 106 cfu/g faeces) than yogurt (5.2 x 105 cfu/g faeces)  (Fig 5).These data indicate that mature Cheddar cheese compares very favourably with freshyogurt as a delivery system for viable probiotic micro-organisms to the gut.  However, therewas no effect on body weight gain or feed conversion efficiency (FCE) in pigs receiving theenterococcal probiotic strain in either Cheddar cheese or yogurt compared with controlanimals.Since probiotic micro-organisms have previously been shown to stimulate/modulate theimmune system, the serum IgG response to the administered probiotic strain was measuredin a subset of the animal population by flow cytometry.  A positive response was found inpigs fed the probiotic strain, while no response was detected in the control animals.However, the mechanisms involved are, as yet, not fully understood.Production of probiotic ingredientIn order to expand the probiotic product portfolio, spray-dried skim-milk powders,containing high numbers of probiotic strains were prepared for subsequent use in thepreparation of probiotic functional foods, such as Cheddar cheese, yogurt and ice cream.Typical counts obtained in these powders at manufacture were 109 - 1010 cfu/g of theprobiotic strain Lb. paracasei NFBC 338 and these levels were maintained followingSurvival of Fargo 688® during cheeseripening1098765432100                     50                         100                          150                         200 250                        300Time (Days)Log CFU/g Control+Fargo 688®Fig. 3.Cheese delivers high numbers of probioticlactobacilli to the porcine GIT*A) Probiotic lactobacilli fed in either 5-mo old Cheddar cheese or fresh yogurt*B) Administered strain recovered from small intestinal contents121086420cheese yogurt                                                          cheese                                yogurtA)  Ingested probiotic                                                                        B)  Probiotic Count in GITLog total cfu ingestedFig. 4.76543210Log cfu/ml small intestine contentsPatentProcess for the manufacture of probiotic cheese. BioResearch Ireland and Forbairt, Patent Application No. 980408.Media UptakeA measure of the interest generated by this project can be gauged by uptake in the media.The following are 3 examples: Trials show how Probiotic Cheese can Boost Immunity to Lots of Nasty Bugs, Irish Times,October 1997.Good Bugs - Cheddar cheese soon might join the ranks of yogurt and some other dairyproducts as food that can deliver a good dose of healthy bacteria, La Crosse Tribune,USA, 9th Sept., 1998Say Cheese, Bioscience, USA, Vol. 48, No. 8, p. 664, August 1998.For further information, please contact Dr. Catherine Stanton or Dr. Paul Ross9Gardiner, G., Stanton, C., Lynch, P.B., Collins, J.K., Fitzgerald, G. and Ross, R.P. (1999).Evaluation of Cheddar Cheese as a Food Carrier for Delivery of a Probiotic Strain to theGastrointestinal Tract.  Journal of Dairy Science, 82: 1379-1387.Gardiner, G., Ross, R.P., Wallace, J.M., Scanlan, F.P., Jagers, P.J.M., Fitzgerald, G.F.,Collins, J.K. and Stanton, C. (1999).  Influence of a Probiotic Adjunct Culture ofEnterococcus faecium on the Quality of Cheddar Cheese.  Journal of Agriculture and FoodChemistry 47: (12) 4907-4916.Stanton, C., Gardiner, G., Meehan, H., Collins, K., Fitzgerald, G., Lynch, P.B. and Ross,R.P. (2000). Market potential for probiotics.  American Journal of Clinical Nutrition (Inpress).Gardiner, G.E., OSullivan, E., Kelly, J., Auty, M.A.E., Fitzgerald, G.F., Collins, J.K., Ross,R.P. and Stanton, C. (2000).  Comparative Survival Rates of Human-Derived ProbioticLactobacillus paracasei and L. salivarius Strains During Heat Treatment and Spray Drying.Applied and Environmental Microbiology 66: (6) 2605-2612.898765432100                5               10              15                20                 25                 30Time (Days)Excretion (Log CFU/g faeces)Fig. 5.Excretion - Cheddar cheese (15 months old)Excretion - yogurt (1 week old)Delivery of viable Fargo 688® to the porcineGIT in cheese or yogurtCheese-fed pigsyieldedsignificantly(P<0.05) highermean dailyprobioticexcretion thanyogurt-fed pigs1110DAIRY PRODUCTS RESEARCH CENTREMoorepark, Fermoy, Co. Cork, IrelandTel: +353 (0) 25 42222 - Fax: +353 (0) 42340E-mail: reception@moorepark.teagasc.ieDr. Catherine Stanton Dr. Paul RossThe Science of Farming and Food

Functional Foods in Relation to Health and Disease (New Probiotic Cheddar Cheese).

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