The Effect of Consumption of Milk Fermented by Lactobacillus Casei Strain Shirota on the Intestinal Microflora and Immune Parame

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The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal micro¯ora and immune parameters in humans

S Spanhaak1, R Havenaar1 and G Schaafsma1

1TNO Nutrition and Food Research Institute, PO Box 360, NL-3700 AJ, Zeist, The Netherlands

Objective: To determine the effect of consumption of milk fermented by Lactobacillus casei strain Shirota (L. casei Shirota) on the composition and metabolic activities of the intestinal micro¯ora, and immune parameters in humans. Subjects: Twenty healthy male subjects aged 40±65 years were selected. Design: A placebo-controlled trial was performed in which 10 subjects were randomly assigned to a control and 10 to a treatment group. During the ®rst and last two weeks of the 8-week study the subjects received a strictly controlled diet without fermented products. The same controlled diet was given during the intermediate 4-week test period but then the treatment group received three times daily 100 ml of fermented milk containing 109 CFU L. casei Shirota=ml, whereas the same amount of unfermented milk was given to the subjects in the control group. Results: In comparison to the control group, the consumption of L. casei Shirota-fermented milk resulted in an increase of the Lactobacillus count in the faeces in which the administered L. casei Shirota was predominant at the level of 107 CFU=g wet faeces. This was associated with a signi®cant increase in Bi®dobacterium counts (P < 0.05). Some shifts in the other bacterial species were found, such as a decreased number of Clostridium; however the differences were not statistically different between the treatment and the control groups. The b-glucuronidase and b-glucosidase activities per 1010 bacteria decreased signi®cantly (P < 0.05) at the second week of the 4-week test period with the consumption of L. casei Shirota-fermented milk. Furthermore, the consumption of the fermented milk product resulted in a slight but signi®cant increase in the moisture content of the faecal samples (P < 0.05). No treatment effects were observed for any of the immune parameters measured (including natural killer (NK) cell activity, phagocytosis and cytokine production). Conclusions: The results suggest that consumption of L. casei Shirota-fermented milk is able to modulate the composition and metabolic activity of the intestinal ¯ora and indicate that L. casei Shirota-fermented milk does not in¯uence the immune system of healthy immunocompetent males. Sponsorship: The study was ®nancially supported by Yakult Honsha Co. Ltd, Tokyo, Japan. Descriptors: fermented milk; immune system; intestinal micro¯ora; lactic acid bacteria; Lactobacillus casei

Introduction et al, 1992), the oral intake of lactobacilli resulted in stimulation of macrophages, lymphocytes and natural There is growing interest in the speci®c health effects of killer (NK) cells, higher production of g-interferon and fermented milk products containing speci®c viable probio- signi®cantly higher secretory IgA responses against patho- tic lactic acid bacteria. It has appeared that many intestinal genic agents (Salmonella, Rotavirus). disturbances may, among other causes, be related to altered Experiments in mice have shown that the growth as well gut mucosal barrier functions and that probiotics offer new as the metastasis of tumours can be inhibited by a Lacto- dietary alternatives for the stabilisation of the intestinal bacillus casei strain (Matsuzaki et al, 1985; Asano et al, micro¯ora (reviewed by Havenaar & Huis in 't Veld, 1992; 1986; Kato et al, 1994). However, the effects are dependent Marteau et al, 1993; Sanders, 1995; Salminen et al, 1996). on the strain of lactobacillus, the method of administration, Consumption of lactobacilli can lead to an increased and the type of tumour cells. Epidemiological research host resistance against pathogens. This may be due to indicates that the consumption of fermented milk products improved competition between bene®cial bacteria, selec- is related to a decreased relative risk of breast cancer in tively stimulated by the probiotic, and pathogenic bacteria women (LeÂ et al, 1986; Van 't Veer et al, 1989). Although or to immunomodulation. The immunomodulating proper- the underlying mechanisms are not known, it is suggested ties of lactobacilli and the possible mechanisms and effects that inactivation or inhibition of the formation of carcino- in relation to intestinal infections have been reviewed by gens in the intestinal tract is induced (Fernandes et al, Havanaar & Spanhaak (1994). In mouse experiments (Per- 1987). Furthermore, enhancement or stimulation of digon et al, 1990; Pouwels et al, 1996) as well as in human immune functions have been described, which may also studies (DeSimone et al, 1988; Isolauri et al, 1991; Kaila contribute to a decrease in the risk of the development or recurrence of cancer (Friend & Shahani, 1984; Aso et al, 1995). Correspondence: Dr R. Havenaar Consensus panels of experts on health attributes of lactic Received 26 October 1997; revised 7 July 1998; accepted 27 July 1998 acid bacteria (Sanders, 1993; LABIP, 1995) concluded that Fermented milk and the intestinal micro¯ora S Spanhaak et al 900 there were promising results related to positive effects of General health parameters the consumption of lactic acid bacteria. Established bene®ts The following general health parameters were measured: were identi®ed on (a) lactose digestion, (b) several types of body weight, body temperature, blood pressure, heart rate. diarrhoeal diseases, (c) reduction of faecal enzymes that Haematological parameters measured included white blood may play a role in colon cancer, and (d) the immune cell, red blood cell and platelet counts; haemoglobin con- system. However, it was also concluded that additional centration; haematocrit (Sysmex K1000-system); the sedi- research is necessary to con®rm these bene®cial effects in mentation rate; and white blood cell differentiation. humans. Biochemical parameters in serum measured included cho- These literature data support the hypothesis that orally lesterol, ASAT, ALAT, g-GT, total protein, albumin, pro- ingested Lactobacillus casei has speci®c health effects tein electrophoresis (albumin, a1-, a2-, b- and g-globulins), related to improvement of the composition and metabolic C-reactive protein (CRP) and a1-antitrypsin (a1-AT). activity of the intestinal micro¯ora and immunomodulation in humans. On the other hand, the probiotic strain should be Faecal micro¯ora safe for repeated human consumption in high numbers. Two grams of fresh faecal samples were collected from the Therefore, the objective of this strictly controlled study was inner part of the stool and were put immediately into pre- to investigate the effect of consumption of a milk product weighed bottles with 17 ml transport medium (TRM). The fermented by L. casei strain Shirota (Yakult1, Yakult samples were weighed and stored at 4CÆ 1C. Within 6 h Honsha Co. Ltd, Tokyo, Japan) in a Western type of diet the samples were homogenised in an anaerobic glove box, in normal healthy subjects in terms of (a) the survival of the pipetted into four marked cryotubes (2 ml), and stored in strain during passage through the gastrointestinal tract, (b) liquid nitrogen. After thawing at 37C in the anaerobic bene®cial changes in the composition and metabolic activ- glove box, 10-fold successive dilutions were made in ity of the intestinal micro¯ora, (c) modulation of immune Peptone Physiological Saline. Aliquots of 0.1 ml of the parameters, and (d) general health parameters and safety appropriate dilution were spread onto the following agar for human consumption. media: Reinforced Clostridial Agar (Oxoid CM151) supplemented with 5 g=l glucose, 75 ml=l sterile horse blood and 75 ml=l (0.4%) China blue (RCB agar) for total Subjects and methods anaerobic bacteria; RCB agar containing 80 mg=l kanamy- Subjects cin and 1 mg=l vancomycin for Bacteroidaceae; Eugon agar Twenty apparently healthy men, 55.8Æ 7.5 (SD) years of (BBL) supplemented with 10 g=l maltose (Merck), 400 ml age were selected for this study. Inclusion criteria were no vegetable juice (Campbell V8) and, after sterilisation, obvious obesity (BMI < 30 kg=m2), normal blood pressure 5 ml=l sterile propionic acid to bring the pH at 6.0Æ 0.2 (WHO criteria), no current medication affecting either the for Bi®dobacterium. These culture media were incubated intestinal ¯ora and=or the immune system and haematolo- anaerobically in gas-tied plastic bags (Merck) at 37C for gical and biochemical parameters. The study was per- 48 to 72 h. formed according to the EU guidelines for Good Clinical Outside the anaerobic glove box, aliquots of 0.09 ml Practice (GCP). Informed consent was obtained from all were spread by spiral plating (Spiral System Instruments, subjects, and the study was approved by the Institute Bethesda, MD, USA) onto the following agar media: External Medical-Ethical Committee. Rogosa agar (Oxoid) for Lactobacillus; LBS agar (Oxoid) containing 10 mg=l vancomycin and 2% lactitol for L. casei Diet and design Shirota (large white colonies); Perfringens agar base During the 8-week study period, 20 subjects, randomly (Oxoid) with 2 vials=l Perfringens SFP selective supple- divided into a treatment group and a control group, ment (Oxoid) and 50 ml=l egg yolk emulsion for Clostri- received a strictly controlled diet with a constant composi- dium; Baird-Parker agar (Oxoid) containing Egg yolk- tion of 2418 kcal (10 MJ), protein 11 en%, fat 28 en%, and Tellurite Emulsion for Staphylococcus; Slanetz and Bartley carbohydrates 61 en%. The study consisted of stabilisation medium (Oxoid) for Enterococcus; Violet Red Bile Glu- (2 weeks), test (4 weeks) and follow-up (2 weeks) periods. cose agar (Oxoid) for Enterobacteriaceae, RCB agar con- During the stabilisation and the follow-up periods, each taining 2 ml=l (1%) tellurite for Bacillus; Oxytetracycline± subject consumed daily 3 6 100 ml sterilised semi- Glucose±Yeast Extract agar (Oxoid) with oxytetracycline skimmed unfermented (Dutch) milk (1.5% fat). During GYE selective supplement for yeasts. These culture media the test period the treatment group received daily were incubated anaerobically (GasPak) or aerobically at 3 6 100 ml L. casei Shirota-fermented milk containing 37C or 24C. After incubation, the speci®c colonies on the 3.1% nonfat dry milk solids, 17% sucrose and ¯avours. selective culture media were counted and the number of The control group received the same volume of unfermen- viable microorganisms per gram faecal sample (CFU=g) ted milk having a similar basic composition as the fermen- were calculated. The mean and standard error per group ted product and packaged in identical bottles. Each batch of were calculated from the log values of the CFU=g. both products was checked at regular intervals for microbial composition. The fermented product contained at least Bacterial enzyme activities 109 CFU L. casei Shirota per ml; the unfermented product Faecal samples for the determination of b-glucosidase, b- was sterile. glucuronidase, urease and tryptophanase were stored at The subjects were housed in the Metabolic Ward of the 720C until the assays were performed. b-Glucosidase TNO Institute during the last three days of every fortnight, activity was determined as follows. Substrate solution (2- starting at the end of the stabilisation period. The subjects nitrophenyl-b-D-glucopyranoside) was added to a homo- had their main meal at the institute each day and received genised suspension of faeces in phosphate-buffered saline the rest of the diet for the next 24 h period (breakfast, lunch, (PBS) pH 6.5 (faecal dilution 1:100). After incubation beverages, snacks and test or control drinks). (20 min, 37C) the enzyme reaction was stopped by the Fermented milk and the intestinal micro¯ora S Spanhaak et al 901 addition of 0.01 mol=l NaOH. After centrifugation (10 min, Immunology 3000 6 g), the o-nitrophenol formed was measured at Lymphocyte subsets: These were determined using fresh 415 nm (Goldin & Gorbach, 1976). whole blood (K3EDTA) and double labelling procedures For b-glucuronidase activity, substrate solution (phe- with ¯uorescein isothiocyanate (FITC)- or phycoerythrin nolphthalein-b-glucuronide) was added to a homogenised (PE)-conjugated antibodies (Becton Dickinson, 1989). The suspension of faeces in PBS pH 6.5 (faecal dilution following combinations of monoclonal antibodies (Becton 1:400). After 15 min incubation at 37C the enzyme Dickinson, San Jose, CA, USA) were used: Leu3 FITC reaction was stopped by the addition of 0.2 mol=l glycerine (CD4)=Leu2 PE (CD8) (T helper=inducer and T suppres- solution (pH 10.4). After centrifugation (10 min, sor=cytotoxic cells); Leu4 FITC (CD3)=HLA-DR PE (T 3000 6 g), the phenolphthalein formed was measured at cells, activated T and B cells); Leu4 FITC 553 nm (Goldin & Gorbach, 1976). (CD3)=Leu11 19 PE (CD16 CD56) (T and NK cells); The tryptophanase activity was measured in faecal Leu18 FITC (CD45RA)=Leu3 PE (CD4) (T naive and T samples diluted with phosphate-buffered saline (PBS, memory cells); Leu1 FITC (CD5)=Leu12 PE (CD19) (T 0.05 mol=l, pH 7.0). To 1 ml diluted sample was added and B cells, B cell subset); Leu4 FITC (CD3)=Leu12 PE 2 ml cold acetone. The mixture was centrifuged and the (CD19) (T and B cells). Flow cytometric analysis was supernatant was discarded. Then 1 ml PBS and 0.05 ml performed on a FACStar PLUS (Becton Dickinson, Moun- toluene were added. The samples were shaken (60 rpm) tain View, CA, USA). for 10 min. A pyridoxal±bovine serum albumin±PBS solution and substrate (tryptophan±PBS) was added to the samples. After 20 min of incubation (37C), colour Natural killer cell (NK) activity: NK activity was mea- reagent (p-dimethylaminobenzaldehyde) was added. This sured using mononuclear cells isolated from heparinised mixture was incubated for 10 min at room temperature blood and 51Cr -labelled target (K562 tumour) cells (MuleÂ and centrifuged. The optical density at 540 nm was & Rosenberg, 1992). Using three different effector:target measured. (E:T) ratios (100:1, 50:1 and 25:1) the lysis of target cells For the determination of urease activity, a test kit with a as represented by the subsequent release of 51Cr was modi®ed manufacturer's protocol was used (urea=ammonia determined as a measure of NK activity. test kit; Boehringer Mannheim, Mannheim, Germany). Urea and a buffer solution (triethanolamine pH 8.0) containing 2-oxoglutarate, glutamate dehydrogenase and Cytokine assays: Interleukin 1b (IL-1b) and 2 (IL-2) and NADH were added to a centrifuged (10 min, 3000g) g-interferon (IFN-g) were measured in culture supernatants faecal suspension. The amount of NADH oxidation was of stimulated (LPS 100 mg=ml (Sigma, St Louis, MO, USA) measured during 10 min at room temperature at 340 nm. All for IL-1b and ConA 20 mg=ml (Sigma) for IL-2 and IFNg) bacterial enzyme activities were expressed in terms of units 6 10 peripheral blood mononuclear cells (10 cells=ml) using (U) per 10 CFU. ELISA kits (IL-1b and IL-2: R&D systems, Minneapolis, MN, USA; IFNg: HBT, Leiden, The Netherlands). Faecal parameters Faecal moisture content was derived from the difference between the faecal dry and wet weights. pH was measured Phagocyte functions: Flow cytometric analyses (FACS- in suspension of the pooled faecal samples. can; Becton Dickinson) of phagocytic capacity and oxida- Intestinal transit time was measured as follows. At tive burst were done in fresh heparinised whole blood, arrival on the ®rst day of each internal period, the subjects using standard kits (Orpegen, Heidelberg, Germany). were given 500 mg carmine red. The time between ingestion and the ®rst appearance of the red colour in the faeces was recorded and taken as the transit time. Neutral sterols Delayed-type hypersensitivity (DTH): To determine (coprostanol, cholesterol, campesterol, b-sitosterol) and effects on the in vivo cellular response at week 9, the bile acids (cholic, lithocholic, deoxycholic, ursodeoxy- DTH reaction after 48 h against eight antigens (Candida, cholic and chenodeoxycholic acid) in faeces were measured Diphtheria, Proteus, Streptococcus, tetanus, Trichophyton, by GLC according to the method of Child et al (1987). tuberculin and glycerine (negative control) was tested using Short-chain fatty acids (acetic, propionic and butyric) the Multitest CMI system (Institut Merieux, Lyon, France). were analysed in faecal water by HPLC using a HPX 87-H column (30 cm 6 7.8 mm, Biorad). Cytotoxicity of faecal water was assessed using a slightly modi®ed version of the Humoral parameters: IgM, IgG, IgA, IgD and IgE and method described by Rafter et al (1987). the complement factors C3, C4 and factor B were measured using a Behring Nephelometric Analyser (Behringwerke AG, Marburg, Germany). Urinary indices Twenty-four-hour urine samples were collected during the periods when the subjects were housed in the metabolic Statistics ward. Spectrophotometric measurement of indican was performed using a colour reaction with thymol and FeCl The statistical signi®cance of differences in changes (Gorter & DeGraaf, 1955). Urine was hydrolysed and between groups was tested by using the non-parametric phenol and p-cresol concentrations were determined by test of Sign±Wilcoxon. This test was performed after taking GLC with ¯ame ionisation detection according to proce- into account initial differences between treatment and dures of BCO laboratories (Breda, The Netherlands). control groups at the end of the stabilisation period. Fermented milk and the intestinal micro¯ora S Spanhaak et al 902 Results Bacteroidaceae, Enterobacteriaceae, Staphylococcus, Sta- phylococcus aureus, Bacillus, Clostridium, Enterococcus General health parameters and yeasts were not signi®cantly different in the treatment Throughout the study, there were no signi®cant changes in group compared to the control group (Table 1). general health parameters such as body weight, blood pressure, heart rate, temperature, haematology and blood Bacterial enzyme activities chemistry in subjects of both the control and treatment Based on enzyme activities calculated per 1010 CFU, groups. between-groups signi®cant changes were observed at week 4 for b-glucuronidase (Table 2, Figure 2; P < 0.05) Faecal micro¯ora and b-glucosidase (Table 2, Figure 2; P < 0.05). Urease and During the test period, the consumption of L. casei Shirota- tryptophanase activity showed no statistically signi®cant fermented milk resulted in a signi®cant increase in the changes. number of the administered L. casei Shirota (P < 0.01), reaching levels of 107 CFU per gram of wet faeces in the Parameters in faeces and urine treatment group compared to the control group (Figure 1). Moisture content was signi®cantly increased (P < 0.05) at Although not statistically signi®cant, a concomitant the end of the test period (Table 2). Faecal pH was increase in the total Lactobacillus count during the test relatively stable throughout the study, varying from 7.0 to period was observed (Table 1). In addition, in week 4 of the 6.8. No statistically signi®cant effects were observed test period a signi®cant increase in the Bi®dobacterium (Table 2). Intestinal transit time tended to decrease in count was observed in the treatment group as compared to both groups. This tendency persisted in the treatment the control group (Table 1; P < 0.05). The numbers of group, resulting in a signi®cant difference (P < 0.05)

Figure 1 Mean numbers and s.e.m. (vertical bars) of Lactobacillus casei Shirota in faecal samples of the treatment (d) and control groups (s). * Signi®cant difference between control and treatment group (P < 0.01).

Table 1 Log numbers of bacteria (meanÆ s.e.m.) per gram faecal sample measured in faecal samples at the end of the stabilisation period (week 2), after 2 and 4 weeks during the test period (week 4 and week 6), and at the end of the follow-up period (week 8)

Control group Treatment group

Parameter Week 2 Week 4 Week 6 Week 8 Week 2 Week 4 Week 6 Week 8

Total anaerobes 9.6Æ 0.4 9.9Æ 0.3 9.9Æ 0.2 9.9Æ 0.3 9.4Æ 0.4 9.9Æ 0.3 9.7Æ 0.3 9.6Æ 0.3 Bacteroidaceae 9.4Æ 0.4 9.6Æ 0.4 9.2Æ 0.4 9.6Æ 0.4 9.2Æ 0.4 9.6Æ 0.5 8.9Æ 0.4 9.5Æ 0.5 Bi®dobacterium 9.1Æ 0.3 9.1Æ 0.6 9.3Æ 0.4 9.3Æ 0.5 8.8Æ 0.5 9.2Æ 0.5a 9.2Æ 0.4 8.9Æ 0.6 Lactobacillus casei Shirota 3.3Æ 2.1 2.9Æ 1.8 4.1Æ 1.8 3.8Æ 1.9 2.0Æ 0.0 7.5Æ 0.5a 7.5Æ 0.6a 2.0Æ 0.0 Lactobacillus total 7.3Æ 0.8 7.1Æ 1.0 6.7Æ 1.2 7.2Æ 0.9 6.8Æ 1.5 7.6Æ 0.7 7.4Æ 0.7 6.9Æ 1.0 Enterococcus 6.2Æ 0.8 5.6Æ 1.2 5.7Æ 0.9 5.2Æ 1.3 5.5Æ 0.8 4.7Æ 1.1 4.3Æ 1.5 4.3Æ 1.4 Clostridium 4.6Æ 1.6 4.5Æ 1.0 3.6Æ 1.8 3.3Æ 2.5 5.2Æ 1.0 4.7Æ 1.0 3.3Æ 2.0 3.7Æ 2.2 Bacillus 3.1Æ 1.1 3.1Æ 1.1 2.6Æ 0.3 2.8Æ 1.1 2.9Æ 1.1 3.6Æ 0.8 3.0Æ 0.5 3.5Æ 0.4 Staphylococcus total 4.2Æ 2.2 2.6Æ 2.0 1.6Æ 1.0 2.4Æ 1.3 4.0Æ 1.8 2.2Æ 0.9 2.0Æ 1.5 1.1Æ 0.9 Staphylococcus aureus 1.0Æ 0.2 1.2Æ 1.0 0.8Æ 0.1 1.0Æ 0.8 1.2Æ 1.2 0.9Æ 0.3 1.1Æ 1.0 0.9Æ 0.6 Enterobacteriaceae 6.6Æ 0.6 6.6Æ 0.9 6.3Æ 1.0 6.4Æ 1.2 6.5Æ 1.5 7.3Æ 0.8 6.6Æ 1.1 6.8Æ 0.9 Yeast 1.9Æ 0.9 2.2Æ 1.3 2.1Æ 1.0 1.6Æ 1.2 1.5Æ 0.8 1.8Æ 1.1 1.4Æ 1.2 1.2Æ 1.1

a Statistically signi®cant difference (P < 0.05) between groups corrected for initial differences. Fermented milk and the intestinal micro¯ora S Spanhaak et al 903 Table 2 Faecal and urinary parameters (meanÆ s.e.m.) measured at the end of the stabilisation period (week 2), after 2 and 4 weeks during the test period (week 4 and week 6), and at the end of the follow-up period (week 8)

Control group Treatment group

Parameter Week 2 Week 4 Week 6 Week 8 Week 2 Week 4 Week 6 Week 8 (Units)

Bacterial enzyme activities Urease 112Æ 43 48Æ 30 34Æ 10 28Æ 7 139Æ 60 32Æ 9 64Æ 14 65Æ 18 (101U=1010CFU) b-Glucuronidase 80Æ 20 45Æ 7 41Æ 6 55Æ 14 167Æ 35 44Æ 6a 72Æ 13 123Æ 24 (1072U=1010CFU) b-Glucosidase 443Æ 117 271Æ 316 215Æ 30 257Æ 50 747Æ 147 230Æ 53a 328Æ 76 548Æ 122 (1072U=1010CFU) Tryptophanase 105Æ 24 61Æ 13 52Æ 8 71Æ 16 155Æ 33 48Æ 7 89Æ 19 131Æ 23 (U=1010CFU)

Faecal parameters Faecal moisture 76Æ 3 76Æ 3 75Æ 2 75Æ 2 72Æ 6 75Æ 3 75Æ 3a 73Æ 4 (%) pH 6.9Æ 0.2 6.6Æ 0.4 6.8Æ 0.3 6.8Æ 0.4 7.0Æ 0.4 6.9Æ 0.2 6.9Æ 0.2 7.0Æ 0.3 Intestinal transit time 45Æ 14 30Æ 16 36Æ 15 37Æ 15 44Æ 14 35Æ 11 29Æ 12 26Æ 18a (h) Cytotoxicity of faecal water 9.6Æ 3.2 11.5Æ 4.3 12.6Æ 4.0 8.8Æ 2.3 13.6Æ 5.2 10.3Æ 2.3 12.4Æ 4.4 8.8Æ 3.0 (% lysis) Coprostanol 60Æ 37 42Æ 18 59Æ 36 66Æ 46 72Æ 30 68Æ 19 64Æ 20 66Æ 20 (mmol=g) Cholesterol 10Æ 17 10Æ 12 6Æ 4 6Æ 4 6Æ 3 5Æ 3 4Æ 2 5Æ 2 (mmol=g) Campesterol 72Æ 115 64Æ 44 53Æ 31 51Æ 22 49Æ 23 50Æ 17 46Æ 21 47Æ 17 (1072 mmol=g) b-Sitosterol 18Æ 22 20Æ 15 12Æ 8 14Æ 8 14Æ 8 14Æ 7 13Æ 6 13Æ 6 (1071 mmol=g) Lithocholic acid 57Æ 21 71Æ 72 61Æ 18 67Æ 20 77Æ 28 67Æ 22 65Æ 24 69Æ 25 (1071 mmol=g) Desoxycholic acid 88Æ 39 105Æ 89 80Æ 28 100Æ 35 111Æ 50 91Æ 43 106Æ 46 103Æ 52 (1071 mmol=g) Chenodeoxycholic acid 13Æ 19 16Æ 23a 14Æ 25 16Æ 19 8Æ 6 6Æ 5 6Æ 3 6Æ 3 (1071 mmol=g) Cholic acid 15Æ 37 18Æ 35 13Æ 28 7Æ 7 8Æ 10 5Æ 9 5Æ 5 5Æ 6 (1071 mmol=g) Ursodesoxycholic acid 35Æ 62 33Æ 54 58Æ 99 51Æ 94 30Æ 64 24Æ 39 13Æ 15 16Æ 18 (1072 mmol=g) Acetic acid 131Æ 49 151Æ 60 127Æ 60 135Æ 51 147Æ 71 94Æ 42a 102Æ 54a 93Æ 42a (mg=100ml) Propionic acid 42Æ 20 59Æ 37 44Æ 21 49Æ 28 42Æ 27 24Æ 17a 30Æ 20a 30Æ 21 (mg=100ml) Butyric acid 52Æ 31 56Æ 31 46Æ 31 46Æ 32 46Æ 37 29Æ 29 35Æ 26 30Æ 22 (mg=100ml)

Urinary indices Indican 39Æ 15 38Æ 10 44Æ 13 38Æ 10 46Æ 15 47Æ 16 44Æ 14 43Æ 13 (mg=ml) Phenol 2.4Æ 1.9 1.0Æ 0.7 1.3Æ 1.1 2.1Æ 1.2 2.4Æ 1.3 1.6Æ 0.9 1.4Æ 1.0 2.2Æ 1.0 (mg=ml) P-Cresol 49Æ 31 38Æ 30 57Æ 27 39Æ 28 62Æ 29 70Æ 44 57Æ 26 52Æ 24 (mg=ml) a Statistically signi®cant difference (P < 0.05) between groups corrected for initial differences.

Figure 2 Mean b-glucuronidase (s, d) and b-glucosidase (n, m) activities and s.e.m. (vertical bars) in faecal samples of the treatment (solid markers) and control groups (open markers) calculated per 1010 CFU. * Signi®cant difference in change of activity between control and treatment group (P < 0.05). between the treatment and control groups at the end of the weeks 4 and 6 (P < 0.01). For butyric acid no statistically follow-up period (Table 2). Faecal concentrations (mmol=g signi®cant changes were found. Cytotoxicity of faecal faeces dry weight) of neutral sterols and bile acids showed water and urinary concentrations of indican, phenol and no signi®cant differences (Table 2). All measured short- P-cresol showed no signi®cant changes when treatment and chain fatty acid (SFCA) (acetic, propionic and butyric) control groups were compared (Table 2). concentrations (mg=100 ml faecal water) showed similar trends, namely a decrease during the test period in the Immune system treatment group (Table 2). When compared between No statistically signi®cant effects were observed in the groups, these decreases were statistically signi®cant for percentages of T cells, CD4 cells, CD8 cells, NK cells acetic acid at weeks 4, 6 and 8 and for propionic acid at and B cells. Furthermore NK activity and production of Fermented milk and the intestinal micro¯ora S Spanhaak et al 904 IFN-g, IL-1b and IL-2 showed no signi®cant difference humans (Marteau & Rambaud, 1993). In view of this, the between the treatment and control groups. Similarly, there present placebo-controlled study in healthy subjects was were no signi®cant differences between the control and performed. the treatment group in the humoral parameters measured Regarding the general health of the subjects, the para- (Table 3). meters measured, such as body weight, blood pressure and No statistically signi®cant treatment effects were blood chemistry, did not reveal any signi®cant changes, observed for phagocytic capacity, oxidative burst (Table indicating that there were no adverse effects in either the 3) and DTH reactions. treatment or the control group throughout the study. The numbers of L. casei Shirota (Figure 1) recovered from the faeces con®rmed the compliance of the subjects to Discussion the study protocol and demonstrated that an adequate The study described in this paper is unique in that it is the percentage of L. casei Shirota survives passage through ®rst double-blind, placebo-controlled study with a commer- the gastrointestinal (GI) tract. Without exception, approxi- cially available probiotic product in healthy humans. mately 107 CFU of this strain per gram faeces were During the last 10 years it has been demonstrated in detected in all samples of the treatment group during the several studies that probiotic strains of lactobacilli, con- test period. After cessation of administration of the fer- sumed via dairy products or given as freeze-dried prepara- mented milk, the numbers of L. casei Shirota returned to tions, may decrease the duration of diarrhoeal disease in pre-treatment levels, indicating that this strain did not children with intestinal infections (particularly with rota- colonise the gut permanently. Similarly, another probiotic virus) and in people with diarrhoea associated with anti- strain of L. casei (later characterised as L. rhamnosus) was biotic treatment (Siitonen et al, 1990; Isolauri et al, 1991, found not to colonise the gut in several studies (Goldin et 1994; Kaila et al, 1992; Sheen et al, 1995). In addition, it al, 1992; Saxelin et al, 1993, 1995). The average total has been demonstrated that probiotic lactobacilli may number of Lactobacillus in the treatment group was not modulate parameters of the immune system (Perdigon et signi®cantly different from that in the control group. How- al, 1990; Sanders, 1993; Kaila et al, 1995; Pouwels et al, ever, in the treatment group the total Lactobacillus popula- 1996). An important question, however, is what effect the tion in the faeces consisted to a large extent of L. casei consumption of probiotic lactobacilli has on intestinal Shirota. ecology of healthy people. In spite of a rather large body The levels of faecal lactobacilli observed in the present of evidence in experimental animals, this question has not study were high as those reported in previous studies (Hill yet been answered, partly because of a lack of well- et al, 1971; Yamagishi et al, 1974; Simon & Gorbach, designed placebo-controlled experiments in healthy 1984; Faassen et al, 1987; Mutai & Tanaka, 1987; Lidbeck,

Table 3 Immunological parameters (meanÆ s.e.m.) measured at the end of the stabilisation period (week 2), after 2 and 4 weeks during the test period (week 4 and week 6), and at the end of the follow-up period (week 8)

Control group Treatment group

Parameter Week 2 Week 4 Week 6 Week 8 Week 2 Week 4 Week 6 Week 8 (Units)

Lymphocyte subsets T helper (CD4) 45Æ 8 46Æ 9 48Æ 9 47Æ 9 44Æ 8 45Æ 6 47Æ 6 45Æ 7 (%) T supp=cyt (CD8) 34Æ 5 33Æ 6 32Æ 6 33Æ 6 36Æ 9 34Æ 9 33Æ 8 34Æ 8 (%) NK (CD16 & 56) 21Æ 8 21Æ 10 18Æ 8 19Æ 10 22Æ 10 19Æ 7 18Æ 8 21Æ 9 (%) pan T (CD5) 70Æ 12 69Æ 12 72Æ 11 71Æ 12 70Æ 10 72Æ 8 72Æ 7 70Æ 9 (%) pan B (CD19) 9Æ 3 8Æ 3 10Æ 4 9Æ 3 8Æ 2 9Æ 2 10Æ 2 9Æ 2 (%) pan T (CD3) 70Æ 11 69Æ 11 71Æ 10 71Æ 12 70Æ 10 71Æ 7 71Æ 7 69Æ 8 (%)

Humoral parameters IgA 32Æ 13 31Æ 12 31Æ 18 32Æ 14 36Æ 12 35Æ 13 36Æ 15 36Æ 14 (1071g=l) IgM 15Æ 5 14Æ 5 15Æ 5 17Æ 5 16Æ 6 15Æ 6 16Æ 6 16Æ 6 (1071g=l) IgG 130Æ 26 129Æ 26 126Æ 24 131Æ 24 146Æ 20 145Æ 22 143Æ 18 144Æ 18 (1071g=l) IgD 24Æ 18 24Æ 16 23Æ 16 29Æ 25 40Æ 36 42Æ 49 47Æ 63 43Æ 59 (U=ml) IgE 42Æ 32 44Æ 34 42Æ 33 40Æ 29 87Æ 76 85Æ 81 85Æ 83 85Æ 82 (U=ml) C3 82Æ 10 81Æ 11 84Æ 14 84Æ 13 83Æ 13 82Æ 13 78Æ 11 80Æ 17 (1072g=l) C4 28Æ 6 27Æ 7 29Æ 8 29Æ 7 27Æ 12 26Æ 11 26Æ 10 27Æ 12 (1072g=l) Factor B 178Æ 41 173Æ 38 185Æ 45 181Æ 44 189Æ 35 182Æ 38 182Æ 33 188Æ 43 (mg=l)

NK activity E:T ratio 25:1 60Æ 6 47Æ 12 50Æ 11 56Æ 8 56Æ 13 51Æ 13 42Æ 14 48Æ 23 (% speci®c activity)

Cytokine assaysa IFNg 176Æ 99 138Æ 71 193Æ 123 193Æ 106 117Æ 48 113Æ 62 108Æ 94 99Æ 53 (10pg=ml) IL-1b 84Æ 26 84Æ 38 92Æ 24 109Æ 41 84Æ 23 73Æ 48 84Æ 35 106Æ 36 (10pg=ml) IL-2 60Æ 30 58Æ 33 50Æ 21 63Æ 40 40Æ 28 48Æ 31 46Æ 22 49Æ 29 (10pg=ml)

Phagocyte functionsb Phagocytosis neutrophils 57Æ 14 56Æ 16 54Æ 15 52Æ 12 55Æ 6 56Æ 11 51Æ 12 47Æ 8 (%) Oxidative burst neutrophils 19Æ 9 24Æ 6 22Æ 8 16Æ 7 20Æ 8 21Æ 7 19Æ 4 15Æ 9 (%)

a For IFNg and IL-2 production mononuclear cells were stimulated with ConA 20mg=ml and for IL-1b production with LPS 100mg=ml during 24h.b The percentage of phagocytosing neutrophils was determined after 2.5min incubation at 37C; the percentage of neutrophils showing an oxidative burst was determined after stimulation with fMLP (5mmol=l) during 10min at 37C. Fermented milk and the intestinal micro¯ora S Spanhaak et al 905 1991). The pre-existing high numbers of indigenous lacto- effects on cytotoxicity of faecal water in the red blood bacilli in the treatment group may have reduced the effects cell lysis assay. of L. casei Shirota administration on the total lactobacillus Studies in animals have demonstrated that oral admin- count. Nevertheless, the number of total Lactobacillus in istration of speci®c strains of lactobacilli may contribute to the treatment group during the test period was higher than an enhancement of both the humoral and the cellular that at the end of the stabilisation and follow-up periods. immune system (Havenaar & Spanhaak, 1994). Previous This observation indicates that the consumption of a high studies with healthy subjects that examined the effects of number of L. casei Shirota increases the total lactobacilli probiotics on the immune system were less well controlled count and does not simply replace the indigenous Lacto- and used high (3 6 1012) or unreported amounts of lactobacillus ¯ora. bacilli per day (DeSimone et al, 1988; Halpern et al, 1991). The administration of L. casei Shirota was associated In the present study no distinct effects on immune with a signi®cant increase in Bi®dobacterium counts, but responses were noted during the consumption of L. casei did not have statistically signi®cant effects on the numbers Shirota-fermented milk. Although differences between the of the other microorganisms. It has been suggested that an present study and those mentioned above, such as the increase in the Bi®dobacterium count may indicate a Lactobacillus strain used, the dose level and the treatment bene®cial effect on the stability of the intestinal ¯ora period, could explain the lack of immune response effects (Mitsuoka, 1990). Since the faecal ¯ora may not accurately in the present study, we think that other factors could also re¯ect the microbial composition in other parts of the GI have played a role. One factor could be the above-men- tract, we cannot exclude the possibility of more pronounced tioned masking effects by already high numbers of Lacto- effects of L. casei Shirota administration on the microbial bacillus in the intestine. Another factor could be that the composition in speci®c parts of the ileum, caecum or colon. selected healthy subjects had an optimal functioning and Synergistic effects of lactobacilli and bi®dobacteria have stable immune system in which clear-cut effects of con- also been observed in vitro in continuous cultures (Cheng sumption of fermented milk were not detectable. In con- & Nagasawa, 1983). trast, Kaila et al (1992) observed the effect of a L. casei With respect to the metabolic activities of the intestinal strain (later characterised as L. rhamnosus) on immune ¯ora, we observed a decrease in the b-glucuronidase and b- functions in rotavirus-infected children. Thus, it may be glucosidase activities, expressed per 1010 bacteria, upon that, with respect to the effects of probiotic lactobacilli on administration of L. casei Shirota. Since these enzymes the immune system, a distinction should be made between may be involved in chemical carcinogenesis (Goldin & healthy, unchallenged subjects and individuals with a Gorbach, 1984), this effect could be viewed as bene®cial. challenged (by infection or otherwise) or suppressed Recent research in patients with super®cial transitional cell immune system. Further studies are needed to establish carcinoma of the bladder indicates that oral administration whether the administration of L. casei Shirota-fermented of L. casei Shirota preparation (3 g per day) signi®cantly milk is able to induce effects on the immune system in reduced the recurrence of this disease after resection with- immunosuppressed or immunocompromised individuals. out side-effects (Aso et al, 1995). Although this observa- While the present study was performed with a rather tion is encouraging, further research is required to limited number of subjects, it is worth noting that a similar investigate the possible bene®ts of lower doses in healthy study in Japan with the same product (Tanaka, 1996) subjects, as used in the present study, before ®nal conclu- showed almost analogous results to the study in the Nether- sions can be drawn. lands, which supports the signi®cance of the effects The observed increase in faecal moisture content (from observed. 72% to 75%) in the treatment group, although small, may For some parameters a change over time was found in be of interest. We can only speculate about the underlying the treatment group as well as the control group. An mechanism. It could re¯ect a decreased intestinal transit in¯uencing factor for this effect may be the short stabilisa- time and=or an osmotic intestinal effect. It is well recog- tion period, which may have been too short for these nised that the formation of short-chain fatty acids by the parameters to reach a steady-state. intestinal ¯ora plays a role in water and electrolyte Test and reference products were identical with respect absorption and stimulates intestinal motility and osmotic to their macronutrient composition; however, their pH pressure (Roberfroid et al, 1995). However, in contrast, values differed (3.5 versus 6.4 respectively). Although we we observed signi®cantly decreased concentrations of cannot completely rule out that this pH difference in¯u- short-chain fatty acids in the faecal samples of the treat- enced the results, we think this is unlikely. We are not ment group. A reduced transit time may be responsible for aware of any data showing an effect of pH on any of the the increase in the faecal moisture content, but the method measured parameters. used was not sensitive enough to detect small changes in We have demonstrated the survival of the ingested L. intestinal transit time. A decrease in intestinal transit time casei Shirota in the GI tract (Figure 1), which was asso- has been recognised as preventing constipation and being ciated with a small increase in the faecal Bi®dobacterium protective with respect to colon cancer risk owing to an count and a small reduction in activity of two bacterial enhancement of the clearance of toxic compounds (Cum- enzymes (b-glucosidase and b-glucuronidase). We think mings et al, 1992). that in healthy subjects with a normal, stable intestinal No signi®cant differences between the treatment and micro¯ora, changes of larger magnitude would not be control groups were noted in the faecal excretion of expected. It could be speculated that the changes observed neutral sterols and bile acids. Secondary bile acids, may provide some additional defence mechanisms particularly deoxycholic acid, may have cytotoxic effects (improvement of mucosal gut barrier, colonisation resis- and increase epithelial cell proliferation and colon cancer tance) in situations where the ecological intestinal balance risk (Jacobs, 1987). The lack of effects on faecal excretion is disturbed by penetration of enteropathogenic microor- of sterols, fatty acids and pH concurs with the absence of ganisms. In addition, the formation of toxic compounds Fermented milk and the intestinal micro¯ora S Spanhaak et al 906 may be in¯uenced, which in the long term may reduce the Hill MJ, Crawther JS, Drasar BS, Hawkswathy, Aries V & Williams REO cancer risk. At this moment, however, there is no direct (1971): Bacteria and etiology of cancer of large bowel. Lancet i, 95±100. evidence in humans for such bene®cial effects. 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