Abdul Manap, Mohd Yazid

Ali, Abdul Manaf

Lim, Long Chang

Mugundun, Shanti

Rezaei, Maryam

Sipat, Abdullah

Universiti Putra Malaysia Institutional Repository

Agricultural SCiencesPharmacokinetics of probiotic microorganisms in the gastro-intestinal tractMohd Yazid Abdul Manap. Abdul Manap All. Abdullah Slp8t. Lim Long Chang. Shant! Mugundun. Maryam RezslcFaculty of Food Science and BiotechnologyUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaTelephone Number of Corresponding Author: 03-li9468401E-mail of Corresponding Authormyaziduifsb.upm.edu.myKey words: pharmacokinetics, probiotic, prebiotic gastro-intestinal tractIntroductionGut health is currently getting much attention among researchers and public. This mainly because many of the physical healthproblems were found to closely connected to the improper functionality of gut. Over the years, study of the microbiologicalsystem in the gut has slowly uncovered the general effects brought to the host. The concept of probiotic appears when effort ismade to modify or alter the composition of the microfloras in the gut to a remedial one by directly introducing the biologicallyimportant beneficial microorganism. Currently, the prebiotic strategy is proposed as an alternative mode to improve the wellbeing of gut. This is the application of indigestible food ingredient that has impact on the metabolism of intestinalmicroorganisms. In the scientific literature, most of the data on prebiotic effect deals with inulin and fructooligosaccharides,and a range of commercial products have been available for many years. Due to the potential synergy between probiotic andprebiotic, foods containing a combination of these ingredients are often referred to as synbiotic. One may also expect theprebiotic to serve as a preliminary growth substance while introducing a special functionality of probiotic into the gut. This isespecially important in view of the weakness of probiotic surviving through gastrointestinal tract to reach an allocated site suchas small and large bowel. On the other hand, the correct pair of probiotic and prebiotic is designed to insure a successfulestablishment of probiotic in the gut among the present of the large microbial assemblage. This is true when the substrateaffinity of the probiotic, for example Bifidobacterium, is varied according to species and even up to the strain level.Materials and MethodsPeptone Yeast Glucose (PY-G) broth previously formulated by Holdeman and Moore (1977) for the cultivation of intestinalbacterial was modified for the screening of inulinase activity from the array of wild type Bifidobacterium pseudocatenulatum.The only changes were the carbon source (glucose) used to oligofructose as pY-O and inulin as PY-I with the addition of agar.Positive results were indicated by the yellow zone appeared on the agar due to bromecresol purple as the acid indication. Thebest strain was selected from this screening test and its maximum specific growth rate (umax) was determined for the purposeof obtaining the suitable dilution rate in the continuous cultivation. The same umax was found in the case of the challengeharmful microorganism employed (in this case: E. coli VIS7). In therapeutic study. two different concentrations ofbifidobacteria were used to challenge the growth of E. coli VIS7 in the continuous system (lOS cfuJmL and 108 cfulmL). Thehigher concentration was prepared via consecutive propagation of bacterial cell in a batch system. The bacteria cells wereharvested by refrigerated centrifugation (8000 rpm for 20 min at 2°C). Pellet cell obtained was suspended in S ml peptonewater (50 % glycerol. v/v) and was further kept in a 12 mL sterile syringe under refrigerated condition (_200q before seedingfor maximum 3 days. The continuous cultivation was initiated when the maximum number of E. coli Vl57 achieved, normallyafter 6 h of incubation. Bifidobacteria was inoculated into the system for at least after 3 resident times of E. coli VIS7cultivation. The antagonistic activity of B. pseudocatenulatum F 117 against E. coli V157 was further evaluated by growthprofile, acidification activity, and organic acid production during interaction. For prophylactic study. the experimentalconditions were the same except that B. pseudocatenulatum F 117 was to be initially cultivated before seeding in E. coli V157.For both experiment, glucose (0.5 %) and oligofructose (0.5 % and 1.0 %) were the main parameter monitored to evaluate forsynergistic effect of bifid abaete ria and prebiotic on antagonism.Results and DiscussionAll B. pseudocatenulatum screened showed excellent growth on pY-O agar with high acid production. The yellow zoneappeared to be large (2:2Smm) and obvious. This observation was true to all wild type strains, B. pseudocatenulatum and alsothe commercial reference strain, B. pseudocatenulatum JCM 1200. B. in/antis ATCe 15698 exhibited moderate growth onPY -0 agar with small (15m~x<25mm) but clear yellow zone. B. breve ATCC 29720 and B. longum 8B536 produced barelyobvious zone (Smrrrsx-cl Smm) on this agar which indicated poor growth. Meanwhile, all B. pseudocatenulatum demonstratedpoor growth on PY-l agar but for strain FI17 and JCM 1200, which displayed moderate growth. No yellow zone «5mm)observed on PY-I agar for the rest of the three reference strains: B. in/antis. B_ breve and B. longum. This implicit in the absentof acidification activity by these strains. Therefore, B. pseudocatenulatum was suspected to possess high activity in utilizingoligofructose and mulin. In that respect, enzyme inulinase must be highly induced for the purpose of breaking down thespecific J3-(2-1) linkages in these molecules.In batch culture, for B. pseudocatenulatum Fl J 7, JIm'was calculated as 0.22 h'!, 0.25 h'! and 0.19 h'! in glucose, oligofructoseand inulin respectively. However, for E. coli V 157, JImwas determined as 1.00 h", 1.10 h-! and 1.20 h'! in the corresponding309UPM Research Report 1997-2000. Vol II.Section 2-Extended Abstractsmedium. 11m in range ofO.to h" to 0.12 h·1 was employed in the continuous interaction of B. pseudocatenulatum FI17 and E.coli V157.In therapeutic study. pH constant at 6.80 was suspected to affect the growth of freshly seeded B. pseudocatenulatum FI17,which unable to proliferate to a higher count. On the contrary, growth stimulation was observed when B. pseudocatenulatumF 117 were introduced into this similar condition except further monitoring of pH. pH lowering was presumably due to increasein organic acids concentration when the population of B. pseudocatenulatum F 117 have considerably elevated. Lactic acid, acentral organic acid in associate with bifidobacteria for the typical bifid shunt though, was not measured. However, batchcultivation with the same growing conditions obser ved an intense amount of lactic acid produced particularly in the initialstage of growth. In summary, under-detection of this compound might be ascribed to poor growth which rendered in the lost ofcompetition for substrates available compared to E. coli \" 157. In addition to that, lactic acid was detected to profusely inducein respond to cell growth for batch cultivation of B. pseudocatenulatum F 117. Consequently, lactic acid might be ofchemically important in terms of growth.By substituting oligofructose as the principal substrate to glucose, two interesting observations were operated concurrently. Amore rapid acidification activity in line with profound growth of B. pseudocatenulatum F 117 was noted. An enhanced growthrate once B. pseudocatenulatum F I 17 was seeded into the continuous flow system was definitely a critical characteristic. It hasfurther approved that oligofructose formerly as the best growth substrate to B. pseudocatenulatum F 117 in batch cultivationperformed equally well to accelerate growth in continuous cultivation. Again, this could be a considerable criterion of prebioticto immediately serve as colonic food for particular probiotic and to the greater extends stimulate its growth.The study with a lower B. pseudocatenulatum F117 dose (I O~cfu/rnl.) in control exhibited a relatively analogous antagonismon E. coli V 157 compared to a higher dose (10' cfu/ml.) used. Besides, the antagonism prevailed could be separated into twointeresting phases. First, while B. pseudocatenulatum F 117 was growing to the maximum count and next was after thismaximum count achieved. It was found that appropriate B. pseudocatenulatum FI17 numbers was rather important, as theantagonistic activity was constantly proved to be higher in the second phase of interaction for either bacterial dose employed.Nevertheless, both B. pseudocatenulatum FI17 doses consumed equally same duration to achieve the maximum growth, thusseemingly implied that lower B. pseudocatenulatum FI17 dose performed comparably excellent to a higher dose. In conditionwith oligofructose fortified medium, no observation with regard to shortening of span for B. pseudocatenulatum F 117 toachieve the maximum growth. Nevertheless, a growth stimulation response was still being detected immediately after seedingof B. pseudocatenulatum F 117 into this environment (the first 6h) compared to control with slower growth rate. Lactateproduction again was stimulated in this medium, as this compound has yet appeared in control at the same time even though acomparable count of B. pseudocatenulatum FI17 was assessed in both conditions. Stimulation in lactate production wastherefore seemed to have a significant utility both for the growth of B. pseudocatenulatum F 117 and also the antagonisticeffect.Current study with higher oligofructose dose did not observe to improve the maximum growth of B. pseudocatenulatum FI17from a lower oligofructose dose assessed. Meanwhile, B. pseudocatenulatum F 117 could hardly raise up to log 9 cfulmL incount as was normally achieved in all other cases. However, the antagonistic effect received by E. coli VI57 was somehowgreatly induced. When the pH declination trend was determined with respect to the lower oligofructose dose used, no apparentjustification could be done. In all, a higher organic acids concentration was much significant to address for such an effect.In prophylactic study, experimental results demonstrated that oligofructose did not significantly increase the resistance of B.pseudocatenulatum FI17 against colonization of incoming E. coli Vl57 when glucose was available as growth substrate.However, E. coli VI57 was unable to sustain in these systems with B. pseudocatenulatum FI17 observed to have higherstability in its viable counts. In PY-G medium, E. coli VI57 has demonstrated sign of resistance after an earlier 12 h of growthreduction. This resistance lasted for 12 h before a more susceptible effect ensued. During this duration, acetic acid was foundto have drastically decreased in concentration. This might infer that E. coli V157 was capable of metabolizing acetic acid asgrowth substrate. However, a further decrease in pH disabled this metabolism to continue. On the other hand, the rate by whichE. coli V 157 eliminated from the system was not enhanced because of prebiotic (PY-0 medium), but a slow and constant rateprevailed. During this period (30 h), acetic acid was also decreasing in concentration, again deducing the metabolism of thiscompound by E. coli VIS7, especially when oligofructose was not a good energy source as discussed earlier on. Lowering ofpH discouraged the growth of E. coli V157, and conversely this condition supported the growth of B. pseudocatenulatumF 117. Nonetheless, the growth of B. pseudocatenulatum F 117 was only limited to log 9 cfulmL in this system.ConclusionsScreening experiment on inulinase activity for oligofructose and inulin has obviously shown that although generally regardedas authentic prebiotic for Bifidobacterium spp., however. the fact could be only reliable to certain strains but not the overallspecies. Wild type B. pseudocatenulatum F 117 was found the best inulin and oligofructose utilizer and was therefore selectedfor advance study. Batch cultivation revealed that B. pseudocatenulatum FI17 was well grown in oligofructose fortifiedmedium instead of glucose or inulm fortified medium. A much higher organic acids production mainly of lactic, acetic andformic acid further approved the unique characteristic of oligofructose and was thus chosen specifically as prebiotic intherapeutic and prophylactic study In therapeutic study, basically, pH and organic acids such as acetic, lactic and formic acidwere the essential inhibitory agent towards the growth of E. coli V157. Most relatively differ and higher was the production oflactic acid from the fermentation of oligofructose to that of glucose. This compound was found to be more important as anacidification agent and also the cytotoxic factor rather than the commonly known acetic acid. Also, at low pH condition, ahigher organic acids concentration could render in a greater inhibitory effect. Nevertheless, these organic compounds wereobserved to profusely produce when the numbers of bifidobacteria was at its maximum. Therefore, the numbers of310Agricultural Sciencesbifidobactcria (cfu/ml.) available for effective interaction was become rather important. In the study of bifidobacteria dose(cfu/rnl.) effect on the growth commencement, the observation was that low initial loads, 105 cfulmL performed comparablyeffective to the higher dose, 10' cfulmL. In both options, the significant antagonism was assessed when the maximum growthof bifidobacteria attained (IOQ cfu/rnl.), And, it happened that both bifidobacteria doses exhibited almost the same duration forthe maximum growth achieved. Nonetheless. when oligofructose was used as the carbon source (rather than glucose), theantagonism displayed much earlier, even before the expected maximum growth due. In the prophylactic study, nevertheless,there wasn't obvious improvement of B. pseudocatenulatum F 117 by oligofructose to resist the exogenously introduced E. coliV 157 as compared to glucose as carbon or energy supply. The pH and organic acids production to this context wereinsufficient to address for such an occurrence. However, in therapeutic and prophylactic study, the antagonism was muchenhanced when the oligofructose concentration used in PY -0 medium increased (from 0.5 % to 1.0 %). A more dramaticantagonistic interaction was illustrated by the elevation of organic acids concentration along with rapid acidification.Nonetheless. too acidic condition was found detrimental to the growth of D. pseudocatenulatum F117.Benefits from the studyThe combination of probiotic (D. pseudocatenulatum F117 and prebiotic (oligofructose) that can fulfilled the criterion ofsynbiotic (synergistic pair) was found. The effect of oligofructose to stimulate the growth of bifidobacteria was furtherexplained in terms of organic acid production. Suitable dose of bifidobacteria for effective therapeutic function could beemployed. Oligo fructose as prebiotic can work to improve the establishment of bifidobacteria community in the allocatedniches of gut. All these studies effectively provide a guideline for future clinical study.Patent(s), if applicableD. pseudocatenulatum F II 7 and oligofructose as "synbiotic pair"Stage of Commercialization, if applicable:NilProject Publications in Refereed JournalsI.Vazid A.M., Ali A.M., Shuhaimi M., KalaivaaniV., Rokiah M.Y. and ReezalA. 2000. Antimicrobialsusceptibilityofbifidobacteria. Letters in Applied Microbiology 31: 57-62.2. K.W.Yap, Rezaei Sabet M, L.C. Lim, Shuhaimi M, Sipat A, Ali AM, Abdul Rahim M, Nur Atiqah AM, Yazid AM. 2003. Bile SaltDeconjugationby Bifidobacteria.Bioscience and Microjlora. (In progress)Project Publications in Conference ProceedingsI. L.C.Um, Yazid AM, A, Ariffand M. Kharidah. 2002. Therapeutic effect ofbifidobacteria against commensal E. coli with prebiotic effectusing continuous flow culture system. In symposium C: Life Science. Malaysian Science and Technology Congress. Pg. 14, 12·14December 2002, Hotel Kuching Hilton,Kuching, Sarawak.Graduate ResearchName of Graduate Research Topic Field of Expertise Degree Awarded Graduation Year(e.g. (or expected)M SC/Ph. D.)Lim Long Chang Synergistic effect of Food M.SC 2004Bifidobacterium and MicroorganismFructooligosaccharides on theantagonism againstEscherichia coliMaryam Rezaei Cholesterol Food PhD. 2001assimilation by BiotechnologyBifidobacterium sppin vitro and in vivostudyIRPA Project number: 09-02-04-0149UPM Research Cluster:AFF311

Pharmacokinetics of probiotic microorganisms in the gastro-intestinal tract

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Pharmacokinetics of probiotic microorganisms in the gastro-intestinal tract

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