Journal of Applied Microbiology 1998, 84, 759–768
Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract W.P. Charteris, P.M. Kelly1, L. Morelli3 and J.K. Collins2 SETTM Consultants Ltd, Douglas, 1National Dairy Products Research Centre, Moorepark, 2Microbiology Department, University College, Cork, Ireland and 3Instituto di Microbiologia, Universita Cattolica del Sacre Coure, Piacenza, Italy 6175/04/97: received 7 April 1997 and accepted 16 September 1997
An in vitro methodology which mimics in vivo human upper gastrointestinal transit was developed. The transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species was determined by exposing washed cell suspensions at 37°C to a simulated gastric juice (pH 2·0), containing pepsin (0·3% w/v) and sodium chloride (0·5% w/v), and a simulated small intestinal juice (pH 8·0), containing pancreatin USP (1 g l−1) and sodium chloride (5 g l−1), and monitoring changes in total viable count periodically. The methodology was also employed to determine the effect of adding milk proteins (1 g l−1), hog gastric mucin (1 g l−1) and soyabean trypsinchymotrypsin inhibitor [SBTCI] (1 g l−1) on transit tolerance. The majority (14 of 15) of isolates lost ×90% viability during simulated gastric transit. Only one isolate, Lactobacillus fermentum KLD, was considered intrinsically resistant. The addition of milk proteins, singly and in combination, generally improved gastric transit tolerance. In this regard, two isolates, Lact. casei 212.3 and Bifidobacterium infantis 25962, exhibited 100% gastric transit tolerance in the presence of milk proteins. In general, the addition of hog gastric mucin did not influence simulated gastric transit tolerance of lactobacilli but tended to increase that of bifidobacteria. However, it increased that of Lact. casei 242 and Lact. salivarius 43338 but diminished that of B. bifidum 2715 and B. animalis Bo. Selected bile salts-resistant isolates were intrinsically tolerant to simulated small intestinal transit. Only Lact. casei F19 and B. adolescentis 15703T showed significant reduction in viability after 240 min. In general, the addition of milk proteins and SBTCI did not affect simulated small intestinal transit tolerance. However, they significantly improved the intrinsic resistance of Lact. casei F19 but diminished that of B. breve 15700T. It is concluded that, whereas the majority of bile salts-resistant lactobacilli and bifidobacteria may be intrinsically sensitive to gastric transit, they are intrinsically resistant to small intestinal transit. In addition, it is postulated that milk proteins and mucin may function as both buffering agents and inhibitors of digestive protease activity in vivo, thereby protecting ingested bacterial strains during upper gastrointestinal transit. W .P . C H AR TE R IS , P . M. KE L LY , L . MO RE L LI AN D J. K. C OL LI N S. 1998.
INTRODUCTION
Manipulation of the human gastrointestinal microbiota is currently being attempted as a means of introducing new Correspondence to: William Charteris, SETTM Consultants Ltd, SETConTM House, 43 Frankfield, Douglas, Cork City, Ireland (e-mail:
[email protected]). © 1998 The Society for Applied Microbiology
micro-organisms into the digestive tract which are beneficial to the human host or are able to bring about advantageous changes to the equilibrium populations and metabolic activities of the indigenous microbiota (Charteris et al. 1997). Micro-organisms that have been used to achieve these goals have been called ‘probiotics’ (Fuller 1991). Lactobacillus and
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Bifidobacterium species constitute a significant proportion of probiotic cultures used in developed countries (Fuller 1992). An essential determinant in the choice of a probiotic microorganism is its ability to reach, survive and persist in the environment in which it is intended to act. Although resistance to human gastric transit has been demonstrated in vivo for potentially probiotic lactic acid bacteria (Kolars et al. 1984; Savaiano et al. 1984; Goldin et al. 1992) and bifidobacteria (Berrada et al. 1991; Bouhnik et al. 1992; Marteau et al. 1992; Pochart et al. 1992) and constitutes an important in vitro selection criterion for probiotic microorganisms (Klaenhammer 1982; Goldin and Gorbach 1989), a satisfactory in vitro method which closely simulates in vivo gastric transit has not been defined. In this regard, the use of HCl-acidified distilled water, broth and buffers has enjoyed widespread use (Hood and Zottola 1988; Clark and Martin 1993; Clark et al. 1993) whereas the use of fresh human gastric secretion has not (Pettersson et al. 1983; Conway et al. 1987; Goldin and Gorbach 1989). However, both of these methods have associated shortcomings in that the former does not accommodate the influence of dietary and non-acid constituents of gastric secretion on gastric transit of ingested micro-organisms during digestion and the latter is restricted by the limited availability of fresh material. Although lactobacilli and bifidobacteria have been isolated from all portions of the human gastrointestinal tract (Molin et al. 1993), the terminal ileum and colon appear to be the preferential sites of colonization of intestinal lactobacilli (Lerche and Reuter 1962; Hirtzmann and Reuter 1963) and bifidobacteria (Fuller 1991), respectively. Unfortunately, most studies on probiotic actions have ignored this basic requirement and, as a result, data on the tolerance of potentially probiotic lactobacilli and bifidobacteria to small intestinal secretions, other than bile, are not yet available (Rambaud et al. 1993; Mikelsaar and Ma¨nder 1994). Moreover, a probiotic micro-organism cannot affect its environment unless its population reaches a certain minimum level which has not yet been exactly determined, but is probably between 106 and 108 cfu g−1 of intestinal contents (Marteau et al. 1993; Rambaud et al. 1993). A number of in vivo studies have provided estimates of the amount of ingested bacteria surviving transit through the upper gastrointestinal tract of adults. Lactobacillus acidophilus ingested in fermented milk has been shown to survive well into the proximal (jejunum) (Robins-Browne and Levine 1981; Clements et al. 1983) and distal (ileum) small intestine (Lindwall and Fonde´n 1984; Pettersson et al. 1985; Alm et al. 1989; Marteau et al. 1992). The ileal recovery of the oral inoculum was 1·3–1·5% (Pettersson et al. 1985; Marteau et al. 1992). Based on the findings of Gilliland et al. (1978), the maximal survival rate of bactobacilli through the entire human gastrointestinal tract has been estimated to be between 2 and 5% assuming an average faecal weight of 150 g d−1
(Marteau et al. 1993). Bifidobacterium species have also been shown to survive well into the distal ileum when ingested in fermented milk and capsules (Hove et al. 1994). In two studies, 23·5% (Pochart et al. 1992) and 37·5% (Marteau et al. 1992) of two strains of bifidobacteria were recovered in the distal ileum after ingestion of fermented milk. Based on these findings, an average survival rate of bifidobacteria through the entire gastrointestinal tract has been estimated to be about 30% (Bouhnik et al. 1992). This value differentiates bifidobacteria from lactobacilli as potentially privileged vectors of probiotic action. In this paper, we present an in vitro methodology which attempts to simulate in vivo gastric and small intestinal transit in the absence of bile salts and peristalsis. The methodology was used (i) to screen a collection of potentially probiotic lactic acid bacteria and bifidobacteria for upper intestinal transit tolerance and (ii) to determine the influence of dietary constituents and mucin on the transit tolerance of these isolates. We do not include the effects of bile salts in the study of small intestinal transit tolerance because bile salts tolerance has traditionally been studied separately and the isolates examined in this study have previously been shown to be resistant to bile salts (Charteris and Kelly 1992; Collins and Thornton 1994). MATERIALS AND METHODS Strains
The origin and characteristics of strains are presented in Table 1. Strains were selected for assay of small intestinal transit tolerance on the basis of their in vitro gastric transit tolerance (Collins and Thornton 1994; this study), bile salts tolerance (Charteris and Kelly 1992; Collins and Thornton 1994) and Caco-2 cell adhesion (Crociani and Ballongue 1994; Crociani et al. 1995; Sarem-Damerdji et al. 1995). Stock cultures of lactobacilli and bifidobacteria were maintained at −20°C on glass beads in MRS (de Man et al. 1960) and TPY (Scardovi 1988) broth containing glycerol (40% w/v) (Jones et al. 1984), respectively. Working cultures were also maintained and subcultured in these media. Prior to assay, strains were serially transferred three times in broth and incubated anaerobically at 37°C for 48 h (AnaerogenTM; Oxoid Unipath Ltd, Basingstoke, Hampshire, UK; BBL GasPakTM, USA). Strain characterization
Differentiation of Bifidobacterium and Lactobacillus isolates was confirmed by the presence of fructose-6-phosphate phosphoketolase in bifidobacterial cell extracts (Trovatelli et al. 1974; Scardovi 1988). The species designation of the isolates received was confirmed by Gram stain, colonial appearance, cell morphology, substrate fermentation (API 50 CHL Sys-
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Table 1 Origin and characteristics of strains
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Other characteristics — –––––––––––––––––––––––––––––––––– Strain Species* Source† Origin Bile tolerance‡ Adhesive capacity§ –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — 242 Lactobacillus acidophilus ACA-DC Traditional Greek cheese Good Good 212.3 Lactobacillus casei ACA-DC Brine of Feta cheese Good Very good GG Lactobacillus rhamnosus Aarla Human adult faeces Good Good F19 Lactobacillus paracasei NIZO Human intestine Good No adherence KLD Lactobacillus fermentum Aarla Human adult faeces Good Poor 43338 Lactobacillus salivarius UCC Human intestine Good Very good 43364 Lactobacillus plantarum UCC Human sigmoid colon Good Good 2715 Bifidobacterium bifidum NCFB NA Good Poor 1453 Bifidobacterium bifidum NCFB NA Good Poor Bo Bifidobacterium animalis NIZO NA Very good Poor 27920 Bifidobacterium infantis NIZO Human infant faeces (Scardovi) Good Poor 25962 Bifidobacterium infantis NIZO Human intestine (Reuter) Good Poor 15700T Bifidobacterium breve NIZO Human intestine (Reuter) Good Very good 15698 Bifidobacterium breve NIZO Human intestine (Reuter) ND ND 15703T Bifidobacterium adolescentis NIZO Human adult intestine Good Poor –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — * Species designation was determined as described in Materials and Methods. † Strains were made available as part of EU FLAIR Project No. 0053 by the following: ACA-DC, Prof. George Kalantzopoulos, Agricultural University, Athens, Greece; Aarla, Prof. Range Fonden, Panova Partner AB, Aarla Group, Stockholm, Sweden; NIZO, Dr Anton Weerkamp, Nethrlands Institute for Dairy Research, Ede, The Netherlands; NCFB, Dr Brian Phillips, National Collection for Food Bacteria, Reading, UK; UCC, Prof. Kevin Collins, National Food Biotechnology Centre, University College, Cork, Ireland. ‡ Bile tolerance was determined by growth assay in fresh human gall bladder bile: good (0·3% v/v), very good (0·5% v/v). § Bacterial adhesion was determined after washing Caco-2 adenocarcinoma cells: poor (³5), good (³10) and very good (×10 epithelial cell−1). NA, Not available; ND, Not determined.
tem; BioMerieux SA, France), enzyme activity (API ZYM System, BioMerieux SA) and product formation (lactate and acetate; Boehringer GmbH, Mannheim, Germany) according to the criteria of Holdeman et al. (1977).
Preparation of simulated gastric and pancreatic juices
Biochemicals
Preparation of milk proteins and mucin
Pepsin (P-7000), pancreatin USP (P-1500), mucin from porcine stomach (M-2378) and soyabean trypsin-chymotrypsin inhibitor (SBTCI; T-9777) were obtained from Sigma Chemical Co. (Basingstoke, Hampshire, UK). Sodium caseinate was obtained from the Kerry Group plc (Listowel, Co. Kerry, Ireland). Whey protein isolate was obtained from Carbery Milk Products Ltd (Ballineen, Co. Cork, Ireland). Maximum recovery diluent and L-cysteine hydrochloride were also obtained from Sigma. Analar sodium chloride and hydrochloric acid were obtained from BDH (Alchem Chemicals Ltd, Little Island, Co. Cork, Ireland).
Sodium caseinate, whey protein isolate and mucin were prepared fresh daily by suspending each singly in sterile saline (0·5% w/v) at a concentration of 1 g l−1. Resuspension of sodium caseinate necessitated heating in excess of 60°C. A milk protein blend was also prepared with a final concentration of 1 g l−1 by mixing sodium caseinate and whey protein isolate solutions in equal volumes.
Simulated gastric and pancreatic juices were prepared fresh daily by suspending pepsin (3 g l−1) or pancreatin USP (1 g l−1) in sterile saline (0·5% w/v) and adjusting the pH to 2·0 or 8·0 with concentrated HCl or 0·1 mol l−1 NaOH using a Model 240 pH meter (Corning Inc., USA).
Preparation of washed cell suspensions
After serial transfer in broth, a 1-ml aliquot was subjected to low speed centrifugation (Jouan Type A-14 bench top
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microfuge) at 5000 g for 5 min and washed three times in phosphate-buffered saline, pH 7·0 (Eisenstadt et al. 1993). The total viable count of the washed cell suspension was determined prior to assay of transit tolerance.
Determination of transit tolerance
The tolerance of washed cell suspensions of Lactobacillus and Bifidobacterium species to simulate gastric and small intestinal transit was determined as follows. To 0·2 ml of washed cell suspension in a 2·0-ml capacity screw-cap microfuge tube (Sarstedt Ltd, Drinagh, Co. Wexford, Ireland) were admixed 1·0 ml of simulated gastric (pH 2·0) or pancreatic (pH 8·0) juice and 0·3 ml NaCl (0·5% w/v). The materials were vortexed using a Model K-550-GE mixer (Scientific Industries Inc., Bohemia, NY, USA) at setting 5 for 10 s and incubated at 37°C in a forced air circulated Gallenkamp incubator (Alchem Chemicals Ltd, Little Island, Co. Cork, Ireland). When assaying gastric transit tolerance, aliquots of 0·1 ml were removed after 1, 90 and 180 min for determination of total viable count. Aliquots were removed after 1 and 240 min for determination of total viable count when assaying for small intestinal transit tolerance.
Determination of transit tolerance in the presence of milk proteins and mucin
The transit tolerance of washed cell suspensions of Lactobacillus and Bifidobacterium species was determined as described above with the exception that 0·3 ml of milk protein solution, singly or in combination, or mucin replaced sodium chloride addition.
Determination of total viable counts
Total viable counts of Lactobacillus species were determined by a pour plate method using MRS agar after serial 10fold dilution in maximum recovery diluent. MRS agar was supplemented with L-cysteine hydrochloride (0·05% w/v) for determination of Bifidobacterium species. Plates were incubated anaerobically at 37°C for 48 h.
RESULTS Effect of simulated transit tolerance on viability
The effect of simulated gastric and small intestinal transit on the viability of selected Lactobacillus and Bifidobacterium species is presented in Table 2. Almost all the isolates examined exhibited complete loss of viability during simulated gastric transit. Only Lact. fermentum KLD exhibited an appreciable level of survival (ca 30% of initial count) and is considered intrinsically tolerant to gastric transit. Lactobacillus casei 212.3 and Lact. rhamnosus GC retained viability during simulated small intestinal transit for up to 4 h and are considered intrinsically tolerant. In contrast, Lact. casei F19 showed a 1·5-log cycle reduction in viability and is considered intrinsically sensitive. Lactobacillus fermentum KLD also showed a reduction in viability but recovered viability after 4 h. Almost all Bifidobacterium strains retained viability during simulated small intestinal transit and are considered intrinsically tolerant. Only Bifidobacterium adolescentis 15703T showed a progressive reduction in viability and is considered intrinsically sensitive. Bifidobacterium breve 15700T also showed a reduction in viability but recovered viability after 4 h. Effect of milk protein and mucin addition on viability during simulated gastric transit
The effect of milk protein addition, singly and in combination, on viability during simulated gastric transit is presented in Table 3. In general, milk protein addition improved simulated gastric transit tolerance. In this regard, two isolates, Lact. casei 212.3 and B. infantis 25962, exhibited complete tolerance to simulated gastric transit in the presence of sodium caseinate, whey protein isolate and a combination thereof. In general, mucin addition did not affect simulated gastric transit tolerance of lactobacilli but increased that of bifidobacteria. However, some exceptions to this general rule were observed. Specifically, mucin addition positively affected Lact. casei 242 and Lact. salivarius 43338 but negatively affected B. bifidum 2715 and B. animalis Bo. Effect of milk protein and soyabean trypsinchymotrypsin inhibitor addition on viability during simulated small intestinal transit
Statistical analysis
Results are expressed as the mean and standard deviation of two determinations. Statistical analysis comprised significance testing of the difference between means using a twotailed Student’s t-test at the levels: 0·05, 0·01 and 0·001.
The effect of milk proteins and SBTCI on viability during simulated small intestinal transit is presented in Table 4. The viability of lactobacilli was unaffected by milk protein and SBTCI addition except for Lact. casei F19. The viability of strain F19 after 1 min was improved by over 33% in the
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Table 2 Effect of simulated gastric and small intestinal transit on viability of Lactobacillus and Bifidobacterium species –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Viable count (log cfu ml−1) during Viable count (log cfu ml−1) during simulated gastric transit tolerance simulated small intestinal transit tolerance — –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — –––––––––––––––––––––––––––––––––––––– Species/strain 0 min 1 min 90 min 180 min 0 min 1 min 240 min –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Lactobacillus 242 6·43 (0·04) 6·25 (0·04)* 5·07 (0·04)*** 3·43 (0·01)*** ND ND ND 212.3 5·00 (0·03) 5·00 (0·04) 3·70 (0·03)*** 3·70 (0·03)*** 8·60 (0·04) 8·60 (0·02) 8·60 (0·02) GG 6·86 (0·04) 6·50 (0·01)** 5·56 (0·01)*** 3·86 (0·01)*** 8·94 (0·04) 8·94 (0·04) 8·91 (0·04) F19 6·00 (0·04) 6·01 (0·04) 4·75 (0·04)*** 4·70 (0·03)*** 7·43 (0·03) 6·01 (0·04)*** 5·88 (0·01)*** KLD 7·82 (0·03) 7·81 (0·04) 7·70 (0·03)*** 7·29 (0·03)** 9·45 (0·04) 9·20 (0·04)* 9·45 (0·04) 43338 7·90 (0·04) 7·78 (0·03) 7·15 (0·04)** 4·90 (0·03)*** ND ND ND 433634 7·05 (0·04) 6·90 (0·03) 5·90 (0·02)*** 5·12 (0·03)*** ND ND ND
Bifidobacterium 2715 8·90 (0·04) 8·74 (0·03)** 7·79 (0·04)** 7·60 (0·03)*** 7·85 (0·04) 7·84 (0·02) 7·84 (0·03) 1453 7·90 (0·04) 7·90 (0·04) 6·60 (0·02)*** 4·90 (0·04)*** 9·48 (0·01) 9·48 (0·04) 9·48 (0·04) Bo 8·10 (0·01) 8·10 (0·01) ND 6·49 (0·01)*** ND ND ND 27920 8·26 (0·03) 8·26 (0·03) 6·95 (0·03)*** 5·26 (0·02)*** 8·48 (0·04) 8·48 (0·04) 8·48 (0·04) 25926 8·81 (0·04) 8·81 (0·03) 7·51 (0·04)*** 5·81 (0·01)*** 9·44 (0·04) 9·43 (0·04) 9·44 (0·02) 15703T 8·56 (0·04) 8·54 (0·01) 5·57 (0·04)*** 5·56 (0·04)*** 9·43 (0·04) 9·38 (0·03) 9·22 (0·04)* 15698 8·38 (0·04) 8·26 (0·03) 5·38 (0·03)*** 5·38 (0·03)*** ND ND ND 15700T 8·12 (0·04) 8·12 (0·01) 5·12 (0·03)*** 5·12 (0·03)*** 9·04 (0·01) 8·74 (0·02)** 9·04 (0·04) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Results are shown as mean (S.D.), n 2. Paired sample, two-tailed Student’s t-test with * P ³ 0·05, ** P ³ 0·01 and *** P ³ 0·001. ND, Not determined.
presence of sodium caseinate and whey protein isolate, an improvement which persisted for up to 4 h. Further improvement in viability (ca 80% of initial count) occurred when milk proteins were added in combination with simulated pancreatic juice. The addition of SBYCI also enhanced small intestinal transit tolerance of strain F19 with similar levels of variability occurring after 4 h. The presence of sodium caseinate and SBTCI during simulated small intestinal transit negatively affected the viability of the two bifidobacteria examined. In contrast, the addition of whey protein isolate did not significantly affect viability. The effects of sodium caseinate were also apparent in the protein blend in the presence of whey protein isolate. DISCUSSION
About 2·5 l of gastric juice is secreted each day having a pH of approximately 2·0 and a salt content of not less than 0·5% w/v (Hill 1990). In contrast, about 0·7 l of pancreatic juice is secreted into the proximal small intestine each day having a pH of about 8·0 and a salt content of not less than 0·5% w/v (Keele and Neil 1965). These secretions present a pH and enzymatic barrier to the survival of ingested micro-organisms
during digestion and act in concert with bile and peristalsis to ensure that the resting small intestine is only heavily colonized in conditions of stasis. An improved in vitro methodology is presented which simulates human gastric and small intestinal transit based on these characteristics which can be employed in the selection of Lactobacillus and Bifidobacterium species for use in probiotic functional foods and nutraceutical preparations. The methodology was employed to study the effects of pepsin, trypsin and chymotrypsin, in the presence and absence of selected milk proteins and hog (as a surrogate for human) gastric mucin and/or SBTCI on bacterial transit tolerance. The intrinsic resistance to acid (and bile) of Lact. delbrueckii subsp. bulgaricus and S. thermophilus is poor (Lindwall and Fonde´n 1984; Conway et al. 1987). Lactobacillus acidophilus and bifidobacteria have been reported to be more resistant but large differences exist between strains (Lindwall and Fonde´n 1984; Sakai et al. 1987; Modler et al. 1990; Berrada et al. 1991; Pochart et al. 1992). In this study, intrinsic resistance to gastric transit tolerance was observed to be a rare probiotic property among the isolates examined and to be influenced by the presence of milk proteins (sodium caseinate and whey protein isolate, singly and in combination)
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Table 3 Effect of milk protein and mucin addition on viability of Lactobacillus and Bifidobacterium species during simulated gastric transit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Viable count (log cfu ml−1) in the presence of Exposure — ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Species/strain time (min) Control NaCas WPI MPB Mucin –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Lactobacillus 242 90 5·07 (0·03) 6·28 (0·04)** 6·25 (0·01)*** 6·18 (0·04)*** 5·60 (0·04)** 180 3·43 (0·01) 6·20 (0·02)** 6·19 (0·03)*** 6·18 (0·01)*** 4·19 (0·03)*** 212.3 90 3·70 (0·03) 5·00 (0·04)*** 5·00 (0·04)*** 5·00 (0·03)*** 3·70 (0·04) 180 3·70 (0·03) 5·00 (0·04)*** 5·00 (0·02)*** 5·00 (0·04)*** 3·70 (0·04) GG 90 5·56 (0·01) 5·48 (0·03) 5·56 (0·04) 6·00 (0·04)** ND 180 3·86 (0·01) 5·26 (0·04)*** 5·56 (0·01)*** 6·09 (0·01)*** ND F19 90 4·75 (0·04) 5·60 (0·04)** 5·04 (0·04)* 5·38 (0·02)** 4·70 (0·04) 180 4·70 (0·03) 5·32 (0·02)** 4·30 (0·04)** 5·20 (0·03)** 4·70 (0·04) KLD 90 7·70 (0·02) 7·81 (0·04) 7·81 (0·03) 7·54 (0·04) 7·58 (0·04) 180 7·29 (0·03) 6·84 (0·04)** 6·48 (0·02)*** 7·15 (0·04) 7·11 (0·04) 43338 90 7·15 (0·04) 7·48 (0·02(** 7·43 (0·02)* 7·62 (0·04)** 6·66 (0·04)** 180 4·90 (0·03) 6·60 (0·04)*** 6·60 (0·04)*** 6·60 (0·02)*** 6·60 (0·04)*** 43364 90 5·90 (0·02) 6·78 (0·04)** 6·67 (0·04)** 6·79 (0·03)*** 6·18 (0·01)** 180 5·12 (0·03) 6·78 (0·02)*** 6·67 (0·02)*** 6·90 (0·02)*** 5·12 (0·02)
Bifidobacterium 2715
90 7·79 (0·04) 8·00 (0·04)* 8·38 (0·04)* 8·08 (0·04)* 8·76 (0·02)*** 180 7·60 (0·03) 6·90 (0·04)** 6·90 (0·01)*** 6·90 (0·02)*** 6·90 (0·02)*** 1453 90 6·60 (0·02) 5·90 (0·02)*** 5·90 (0·04)** 5·90 (0·04)** 5·90 (0·04)** 180 4·92 (0·04) 5·90 (0·04)*** 5·90 (0·04)*** 5·90 (0·01)*** 5·90 (0·01)*** Bo 90 ND ND ND ND ND 180 6·49 (0·01) 6·65 (0·04)* 6·58 (0·04)** 6·87 (0·03)** 6·10 (0·04)** 27920 90 6·97 (0·03) 7·26 (0·03)** 6·26 (0·03)** 6·26 (0·04)** ND 180 5·26 (0·02) 6·26 (0·03) 6·26 (0·04) 6·26 (0·04) ND 25962 90 7·51 (0·04) 8·81 (0·t01)*** 8·81 (0·04)*** 8·81 (0·02)*** 8·65 (0·02)*** 180 5·81 (0·01) 8·81 (0·04)*** 8·81 (0·04)*** *·76 (0·03)*** 8·08 (0·02)*** 15703T 90 5·57 (0·04) 7·86 (0·02)*** 7·70 (0·02)*** 7·90 (0·04)*** 8·52 (0·04)*** 180 5·56 (0·04) 7·70 (0·04)*** 7·70 (0·04)*** 7·70 (0·02)*** 6·56 (0·01)*** 15698 90 5·38 (0·03) 6·38 (0·04)** 6·38 (0·03)*** 6·38 (0·03)*** 6·38 (0·03)*** 180 5·38 (0·03) 6·38 (0·04)*** 6·38 (0·02)*** 6·38 (0·04)** 6·38 (0·03)*** 15700T 90 5·12 (0·03) 6·12 (0·04)** 6·12 (0·04)** 6·12 (0·02)*** 6·12 (0·04)** 180 5·12 (0·03) 6·12 (0·03)*** 6·12 (0·04)** 6·12 (0·04)** 6·12 (0·02)*** –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Results are shown as mean (S.D.), n 2. Paired sample, two-tailed Student’s t-test with * P ³ 0·05, ** P ³ 0·01 and *** P ³ 0·001. ND, Not determined; NaCas, sodium caseinate; WPI, whey pretein isolate; MPB, milk protein blend.
and mucin. Only Lact. fermentum KLD (a human enteric isolate, exhibiting adhesion to intestinal epithilia, inhibition of intestinal pathogens, bile salts resistance, and used commercially in Sweden for fermented milk manufacture) could be regarded as being intrinsically resistant to human gastric transit and as reaching the upper small intestine in high numbers after ingestion in the fasting state. The viability of strain KLD during simulated gastric transit tended to diminish in the presence of milk protein (from 29·7 to 20·9% of initial count) and mucin (from 21·9 to 20·3% of initial
count) but the reduction was not statistically significant for mucin. Lactobacillus rhamnosus GG exhibited limited tolerance to simulated gastric transit with ³0·1% of exposed cells surviving beyond 1·5 h. This observation confirms those of Goldin and Gorbach (1989) who observed that strain GG decreased from 109 to 103 after 2 h at pH 1–2 in the human stomach. Indeed, they also observed that strain GG decreased from 108 to ³106 after 0·5 h at pH 2·5 in normal human gastric juice.
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Table 4 Effect of milk protein and soybean trypsin-chymotrypsin addition on viability of Lactobacillus and Bifidobacterium species during simulated small intestinal transit –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Viable count (log cfu ml−1) in the presence of Exposure –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Species/strain time (min) Control NaCas WPI MPB SBTCI –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Lactobacillus F19 1 6·01 (0·04) 7·00 (0·04)** 7·04 (0·04)** 7·36 (0·02)*** 7·13 (0·02)** 240 5·88 (0·01) 7·06 (0·02)** 7·00 (0·01)** 7·33 (0·04)*** 7·32 (0·04)*** Bifidobacterium 15703T 1 9·38 (0·03) 8·96 (0·02)** 9·33 (0·04) 9·36 (0·04) 9·33 (0·02) 240 9·22 (0·04) 8·96 (0·01)* 9·15 (0·04) 9·34 (0·03) 9·00 (0·04)* 15700T 1 8·74 (0·02) 9·04 (0·04)** 8·59 (0·04)* 9·00 (0·04)* 9·04 (0·02)** 240 9·04 (0·04) 8·26 (0·04)** 9·04 (0·04) 8·90 (0·01)* 6·89 (0·04)*** –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Results are shown as mean (S.D.), n 2. Paired sample, two-tailed Student’s t-test with * P ³ 0·05, ** P ³ 0·01 and *** P ³ 0·001. Strains 212.3, GG, KLD, 2715, 1453, 27920 and 25962 did not exhibit a reduction in viability at 1 or 240 min (data not shown). Strains 242, 43338, 43364, Bo and 15698 not examined. NaCas, Sodium caseinate; WPI whey protein isolate; MPB, milk protein blend; SBTCI, soybean trypsin-chymotrypsin inhibitor.
The presence of milk proteins, singly and in combination, exerted a major effect on the gastric tolerance of some strains but not others. In this regard, Lact. casei 212.3 and B. bifidum 25962 were capable of undiminished survival during simulated gastric transit in the presence of sodium caseinate, whey protein isolate and a combination thereof. These data indicate that some strains of Lactobacillus and Bifidobacterium species may survive passage through the human stomach, particularly when ingested with milk products or milk protein-based foodstuffs. Survival of lactic acid bacteria in human gastric juice adjusted to low pH has been previously shown to be enhanced by the addition of skim milk (Conway et al. 1987). The addition of porcine gastric mucin to simulated gastric juice increased the gastric transit tolerance of a majority (seven of 15) of strains examined. However, two strains were negatively affected by mucin, B. bifidum 2715 and B. animalis Bo. The mode of action underpinning the positive effect of mucin is considered to involve protease inhibition although it is not clear whether native mucins in gastric (and intestinal) secretions exhibit this property (Allen 1981). The ability of mucin to resist protease activity may be attributed to the structural characteristics of mucin molecules, in particular the presence of a dense coat of sulphated oligosaccharide chains and covalently bound fatty acids (Slomiany et al. 1996). Furthermore, the ability of mucin to protect some strains of probiotic micro-organisms against protease activity may also involve the acquisition of a surface coating of mucin (Tunnicliff 1940) thus providing a shield against proteolytic activity. Sims (1964) has observed enhanced survival of lactobacilli in protease-free hog gastric mucin solutions. When gastric transit tolerance was determined using fresh
human gastric juice and compared with the results presented in this study, there was very good agreement (data not shown). Of the strains examined disagreement was only found for B. animalis Bo, which exhibited much greater tolerance to human gastric juice than towards simulated gastric juice containing milk protein or mucin. It is unclear why this occurred. The small intestinal transit tolerance of bile salts-resistant lactobacilli and bifidobacteria was found to be strain-dependent. The majority of strains were intrinsically resistant to simulated pancreatic juice and showed no reduction in viability for up to 4 h. However, a minority of strains were sensitive and could be divided into two groups. The first was characterized by complete or progressive loss of viability during exposure and was represented by strain B. adolescentis 15703T. The second was characterized by a rapid reduction in viability on exposure followed by recovery of viability during prolonged exposure and was represented by Lact. fermentum KLD and B. breve 15700T. The viability of a major of lactobacilli and bifidobacteria in simulated pancreatic juice was unaffected by milk protein and SBTCI addition except for Lact. casei F19, B. breve 15700T and B. adolescentis 15703T. Sodium caseinate improved the viability of strains F19 (33% of initial count) and 15700T (50% of initial count) after 1 min but significantly reduced viability after 4 h. Whey protein isolate addition did not produce similar affects, which was also apparent when combined with sodium caseinate for strains F19 and 15700T. The addition of SBTCI decreased the simulated small intestinal transit tolerance of strains 15700T and 15703T. On the basis of this and other studies, many of the strains
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examined here may be regarded as suitable candidate microorganisms for functional food product development and in vivo studies in humans to determine potential health benefits. Strains GG and KLD have already been commercialized and their potential health benefits supported by numerous in vivo and in vitro studies. In this regard, Lact. rhamnosus GG has been shown to possess gastric transit tolerance (Gorbach and Goldin 1989; Goldin et al. 1992; Charteris et al. 1994, 1996), bile salts tolerance (Gorbach and Goldin 1989) and the ability to adhere to human ileal (Silva et al. 1987) and colon adenocarcinoma cell lines in culture (Elo et al. 1991; Chauvie`re et al. 1992). These observations coupled with its intrinsic resistance to intestinal proteases shown in this study suggest that this strain may become established in the human host at this site (Goldin and Gorbach 1987; Goldin et al. 1992; Ling et al. 1992). Lactobacillus fermentum KLD, a human enteric isolate, has been shown to adhere to intestinal epithilia, inhibit certain pathogens and possess bile salts resistance (Department of General & Marine Microbiology, University of Go¨teborg, Sweden). These observations coupled with its intrinsic in vitro gastric transit tolerance and small intestinal transit tolerance (this study) indicate that this strain may colonize the upper human gastrointestinal tract. Lactobacillus casei 212.3 and Lact. plantarum 43364 were also considered resistant to upper gastrointestinal transit in the presence of milk proteins. This observation, coupled with their good human gastric juice and gall-bladder bile tolerance (Collins and Thornton 1994) and very good Caco-2 cell adhesion capacity (Thornton et al. 1993; Sarem-Damerdji et al. 1995), suggests that they may also colonize the human intestine. Furthermore, the sensorial and textural characteristics of bioyoghurt produced using these cultures are commercially promising (Mortberg-Backlund and Fonde´n 1994). ACKNOWLEDGEMENTS
The authors acknowledge funding under the EU FLAIR Programme [project no. AGRF-CT91-0053], the support of Drs Hoestra and Cornelese on behalf of the Commission and Mr Eddie O’Neill and Ms Teresa O’Brien for technical assistance. REFERENCES Allen, A. (1981) Structure and function of gastrointestinal mucus. In Physiology of the Gastrointestinal Tract ed. Johnson, L.R. Chap. 20, pp. 617–639. New York: Raven Press. Alm, L., Leijonmarck, C-E., Persson, A-K. and Midtvedt, T. (1989) Survival of lactobacilli during digestion: an in vitro and in vivo study. In The Regulatory and Protective Role of the Normal Microflora ed. Grubb, R., Midtvedt, T. and Norin, E. Chap. 18, pp. 293–297. London: Macmillan Press.
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